Novel method for down-regulation of amyloid

ABSTRACT

Disclosed are novel methods for combatting diseases characterized by deposition of amyloid. The methods generally rely on immunization against amyloid precursor protien (APP) or beta amyloid (Aβ). Immunization is preferably effected by administration of analogues of autologous APP or Aβ, said analogues being capable of inducing antibody production against the autologous amyloidogenic polypeptides. Especially preferred as an immunogen is autologous Aβ which has been modified by introduction of one single or a few foreign, immunodominant and promiscuous T-cell epitopes. Also disclosed are nucleic acid vaccination against APP or Aβ and vaccination using live vaccines as well as methods and means useful for the vaccination. Such methods and means include methods for the preparation of analogues and pharmaceutical formulations, as well as nucleic acid fragments, vectors, transformed cells, polypeptides and pharmaceutical formulations.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of InternationalPatent Application PCT/DK02/00547 filed Aug. 20, 2002 and published asWO 03/015812 on Feb. 27, 2003, which claims priority from each of:Danish Provisional Patent Applications PA 2001 01231 filed Sugust 20,2001, and PA 2002 00558 filed Apr. 16, 2002 and U.S. Provisional PatentApplication 60/337,543 filed Oct. 22, 2001 and Ser. No. 60/373,027 filedApr. 16, 2002.

The above-referenced patents and applications, and each document citedin this text (“application cited documents”) and each document cited orreferenced in each of the application cited documents, and anymanufacturer's specifications or instructions for any products mentionedin this text and in any document incorporated into this text, are herebyincorporated herein by reference; and, technology in each of thedocuments incorporated herein by reference can be used in the practiceof this invention.

It is noted that in this disclosure, terms such as “comprises”,“comprised”, “comprising”, “contains”, “containing” and the like canhave the meaning attributed to them in U.S. Patent law; e.g., they canmean “includes”, “included”, “including” and the like. Terms such as“consisting essentially of” and “consists essentially of” have themeaning attributed to them in U.S. Patent law, e.g., they allow for theinclusion of additional ingredients or steps that do not detract fromthe novel or basic characteristics of the invention, i.e., they excludeadditional unrecited ingredients or steps that detract from novel orbasic characteristics of the invention, and they exclude ingredients orsteps of the prior art, such as documents in the art that are citedherein or are incorporated by reference herein, especially as it is agoal of this document to define embodiments that are patentable, e.g.,novel, nonobvious, inventive, over the prior art, e.g., over documentscited herein or incorporated by reference herein. And, the terms“consists of” and “consisting of” have the meaning ascribed to them inU.S. Patent law; namely, that these terms are closed ended.

FIELD OF THE INVENTION

The present invention relates to improvements in therapy and preventionof Alzheimer's disease (AD) and other diseases characterized bydeposition of amyloid, e.g. characterized by amyloid deposits in thecentral nervous system (CNS). More specifically, the present inventionprovides a method for down-regulating (undesired) deposits of amyloid byenabling the production of antibodies against a relevant protein (APP orAβ) or components thereof in subjects suffering from or in danger ofsuffering from diseases having a pathology involving amyloid deposition.The invention also provides for methods of producing polypeptides usefulin this method as well as for the modified polypeptides as such. Alsoencompassed by the present invention are nucleic acid fragments encodingthe modified polypeptides as well as vectors incorporating these nucleicacid fragments and host cells and cell lines transformed therewith.Finally, the present invention also provides for a new type of conjugatepeptide immunogen.

BACKGROUND OF THE INVENTION

Amyloidosis is the extracellular deposition of insoluble protein fibrilsleading to tissue damage and disease (Pepys, 1996; Tan et al., 1995;Kelly, 1996). The fibrils form when normally soluble proteins andpeptides self-associate in an abnormal manner (Kelly, 1997).

Amyloid is associated with serious diseases including systemicamyloidosis, AD, maturity onset diabetes, Parkinson's disease,Huntington's disease, fronto-temporal dementia and the prion-relatedtransmissible spongiform encephalopathies (kuru and Creutzfeldt-Jacobdisease in humans and scrapie and BSE in sheep and cattle, respectively)and the amyloid plaque formation in for instance Alzheimer's seems to beclosely associated with the progression of human disease. In animalmodels over-expression, or the expression of modified forms, of proteinsfound in deposits, like the β-amyloid protein, has been shown to inducevarious symptoms of disease, e.g. Alzheimer's-like symptoms. There is nospecific treatment for amyloid deposition and these diseases are usuallyfatal.

The subunits of amyloid fibrils may be wild-type, variant or truncatedproteins, and similar fibrils can be formed in vitro from oligopeptidesand denatured proteins (Bradbury et al., 1960; Filshie et al., 1964;Burke & Rougvie, 1972). The nature of the polypeptide component of thefibrils defines the character of the amyloidosis. Despite largedifferences in the size, native structure and function of amyloidproteins, all amyloid fibrils are of indeterminate length, unbranched,70 to 120 Å in diameter, and display characteristic staining with CongoRed (Pepys, 1996). They are characteristic of a cross-β structure(Pauling & Corey, 1951) in which the polypeptide chain is organized inβ-sheets. Although the amyloid proteins have very different precursorstructures, they can all undergo a structural conversion, perhaps alonga similar pathway, to a misfolded form that is the building block of theβ-sheet helix protofilament.

This distinctive fibre pattern led to the amyloidoses being called theβ-fibrilloses (Glenner, 1980a,b), and the fibril protein of AD was namedthe β-protein before its secondary structure was known (Glenner & Wong,1984). The characteristic cross-β diffraction pattern, together with thefibril appearance and tinctorial properties are now the accepteddiagnostic hallmarks of amyloid, and suggest that the fibrils, althoughformed from quite different protein precursors, share a degree ofstructural similarity and comprise a structural superfamily,irrespective of the nature of their precursor proteins (Sunde M, SerpellL C, Bartlam M, Fraser P E, Pepys M B, Blake C C F J Mol Biol 1997 Oct.31; 273(3):729-739).

One of the most widespread and well-known diseases where amyloiddeposits in the central nervus system are suggested to have a centralrole in the progression of the disease, is AD.

AD

Alzheimer's disease (AD) is an irreversible, progressive brain disorderthat occurs gradually and results in memory loss, behavibural andpersonality changes, and a decline in mental abilities. These losses arerelated to the death of brain cells and the breakdown of the connectionsbetween them. The course of this disease varies from person to person,as does the rate of decline. On average, AD patients live for 8 to 10years after they are diagnosed, though the disease can last for up to 20years.

AD advances by stages, from early, mild forgetfulness to a severe lossof mental function. This loss is known as dementia. In most people withAD, symptoms first appear after the age of 60, but earlier onsets arenot infrequent. The earliest symptoms often include loss of recentmemory, faulty judgment, and changes in personality. Often, people inthe initial stages of AD think less clearly and forget the names offamiliar people and common objects. Later in the disease, they mayforget how to do even simple tasks. Eventually, people with AD lose allreasoning ability and become dependent on other people for theireveryday care. Ultimately, the disease becomes so debilitating thatpatients are bedridden and likely to develop other ill-nesses andinfections. Most commonly, people with AD die from pneumonia.

Although the risk of developing AD increases with age, AD and dementiasymptoms are not a part of normal aging. AD and other dementingdisorders are caused by diseases that affect the brain. In normal aging,nerve cells in the brain are not lost in large numbers. In contrast, ADdisrupts three key processes: Nerve cell communication, metabolism, andrepair. This disruption ultimately causes many nerve cells to stopfunctioning, lose connections with other nerve cells, and die.

At first, AD destroys neurons in parts of the brain that control memory,especially in the hippocampus and related structures. As nerve cells inthe hippocampus stop functioning properly, short-term memory fails, andoften, a person's ability to do easy and familiar tasks begins todecline. AD also attacks the cerebral cortex, particularly the areasresponsible for language and reasoning. Eventually, many other areas ofthe brain are involved, all these brain regions atrophy (shrink), andthe AD patient becomes bedridden, incontinent, totally helpless, andunresponsive to the outside world (source: National Institute on AgingProgress Report on Alzheimer's Disease, 1999).

The Impact of AD

AD is the most common cause of dementia among people age 65 and older.It presents a major health problem because of its enormous impact onindividuals, families, the health care system, and society as a whole.Scientists estimate that up to 4 million people currently suffer fromthe disease, and the prevalence doubles every 5 years beyond age 65. Itis also estimated that approximately 360,000 new cases (incidence) willoccur each year, though this number will increase as the population ages(Brookmeyer et al., 1998).

AD puts a heavy economic burden on society. A recent study in the UnitedStates estimated that the annual cost of caring for one AD patient is$18,408 for a patient with mild AD, $30,096 for a patient with moderateAD, and $36,132 for a patient with severe AD. The annual national costof caring for AD patients in the US is estimated to be slightly over $50billion (Leon et al., 1998).

Approximately 4 million Americans are 85 or older, and in mostindustrialized countries, this age group is one of the fastest growingsegments of the population. It is estimated that this group will numbernearly 8.5 million by the year 2030 in the US; some experts who studypopulation trends suggest that the number could be even greater. As moreand more people live longer, the number of people affected by diseasesof aging, including AD, will continue to grow. For example, some studiesshow that nearly half of all people age 85 and older have some form ofdementia. (National Institute on Aging Progress Report on Alzheimer'sDisease, 1999)

The Main Characteristics of AD

Two abnormal structures in the brain are the hallmarks of AD: amyloidplaques and neurofibrillary tangles (NFT). Plaques are dense, largelyinsoluble deposits of protein and cellular material outside and aroundthe brain's neurons. Tangles are insoluble twisted fibres that build upinside neurons.

Two types of AD exist: familial AD (FAD), which follows a certainpattern of inheritance, and sporadic AD, where no obvious pattern ofinheritance is seen. Because of differences in the age at onset, AD isfurther described as early-onset (occurring in people younger than 65)or late-onset (occurring in those 65 and older). Early-onset AD is rare(about 10 percent of cases) and generally affects people aged 30 to 60.Some forms of early-onset AD are inherited and run in families.Early-onset AD also often progresses faster than the more common,late-onset form.

All FADs known so far have an early onset, and as many as 50 percent of.FAD cases are now known to be caused by defects in three genes locatedon three different chromosomes. These are mutations in the APP gene onchromosome 21; mutations in a gene on chromosome 14, called presenilin1; and mutations in a gene on chromosome 1, called presenilin 2. Thereis as yet no evidence, however, that any of these mutations play a majorrole in the more common, sporadic or non-familial form of late-onset AD.(National Institute on Aging Progress Report on Alzheimer's Disease,1999)

Amyloid Plaques

In AD, amyloid plaques develop first in areas of the brain used formemory and other cognitive functions. They consist of largely insolubledeposits of beta amyloid (hereinafter designated Aβ)—a protein fragmentof a larger protein called amyloid precursor protein (APP, the aminoacid sequence of which is set forth in SEQ ID NO: 2)—intermingled withportions of neurons and with non-nerve cells such as microglia andastrocytes. It is not known whether amyloid plaques Ithemselvesconstitute the main cause of AD or whether they are a by-product of theAD process. Certainly, changes in the APP protein can cause AD, as shownin the inherited form of AD caused by mutations in the APP gene, and Aβplaque formation seems to be closely associated with the progression ofthe human disease (Lippa C. F. et al. 1998).

APP

APP is one of many proteins that are associated with cell membranes.After it is made, APP becomes embedded in the nerve cell's membrane,partly inside and partly outside the cell. Recent studies usingtransgenic mice demonstrate that APP appears to play an important rolein the growth and survival of neurons. For example, certain forms andamounts of APP may protect neurons against both short- and long-termdamage and may render damaged neurons better able to repair themselvesand help parts of neurons grow after brain injury.

While APP is embedded in the cell membrane, proteases act on particularsites in APP, cleaving it into protein fragments. One protease helpscleave APP to form Aβ, and another protease cleaves APP in the middle ofthe amyloid fragment so that Aβ cannot be formed. The Aβ formed is oftwo different lengths, a shorter 40 (or 41) amino acids Aβ that isrelatively soluble and aggregates slowly, and a slightly longer, 42amino acids “sticky” Aβ that rapidly forms insoluble clumps. While Aβ isbeing formed, it is not yet known exactly how it moves through or aroundnerve cells. In the final stages of this process, the “sticky” Aβaggregates into long filaments outside the cell and, along withfragments of dead and dying neurons and the microglia and astrocytes,forms the plaques that are characteristic of AD in brain tissue.

Some evidence exists-that the mutations in APP render more likely thatAβ will be snipped out of the APP precursor, thus causing either moretotal Aβ or relatively more of the “sticky” form to be made. It alsoappears that mutations in the presenilin genes may contribute to thedegeneration of neurons in at least two ways: By modifying Aβ productionor by triggering the death of cells more directly. Other researcherssuggest that mutated presenilins 1 and 2 may be involved in acceleratingthe pace of apoptosis.

It is to be expected that as the disease progresses, more and moreplaques will be formed, filling more and more of the brain. Studiessuggest that it may be that the Aβ is aggregating and disaggregating atthe same time, in a sort of dynamic equilibrium. This raises the hopethat it may be possible to break down the plaques even after they haveformed. (National Institute on Aging Progress Report on Alzheimer'sDisease, 1999).

It is believed that Aβ is toxic to neurons. In tissue culture studies,researchers observed an increase in death of hippocampal neurons cellsengineered to over-express mutated forms of human APP compared toneurons over-expressing the normal human APP (Luo et al., 1999).

Furthermore, overexpression or the expression of modified forms of theAβ protein has in animal models been demonstrated to induceAlzheimer-like symptoms, (Hsiao K. et al., 1998)

Given that increased Aβ generation, its aggregation into plaques, andthe resulting neurotoxicity may lead to AD, it is of therapeuticinterest to investigate conditions under which Aβ aggregation intoplaques might be slowed down or even blocked.

Presenilins

Mutations in presenilin-1 (S-180) account for almost 50% of all cases ofearly-onset familial AD (FAD). Around 30 mutations have been identifiedthat give rise to AD. The onset of AD varies with the mutations.Mutations in presenilin-2 account for a much smaller part of the casesof FAD, but is still a significant factor. It is not known whetherpresenilins are involved in sporadic non-familial AD. The function ofthe presenilins is not known, but they appear to be involved in theprocessing of APP to give AP-42 (the longer stickier form of thepeptide, SEQ ID NO: 2, residues 673-714), since AD patients withpresenilin mutations have increased levels of this peptide. It isunclear whether the presenilins also have a role in causing thegeneration of NFT's. Some suggest that presenilins could also have amore direct role in the degeneration of neurons and neuron death.Presenilin-1 is located at chromosome 14 while presenilin-2 is linked tochromosome 1. If a person harbours a mutated version of just one ofthese genes he or she is almost certain to develop early onset AD.

There is some uncertainty to whether presenilin-1 is identical to thehypothetical gamma-secretase involved in the processing of APP (Naruseet al., 1998).

Apolipoprotein E

Apolipoprotein E is usually associated with cholesterol, but is alsofound in plaques and-tangles of AD brains. While alleles 1-3 do not seemto be involved in AD there is a significant correlation between thepresence of the APOE-ε4 allele and development of late AD (Strittmatteret al., 1993). It is, however, a risk factor and not a direct cause asis the case for the presenilin and APP mutations and it is not limitedto familial AD.

The ways in which the ApoE ε4 protein increases the likelihood ofdeveloping AD are not known with certainty, but one possible theory isthat it facilitates Aβ buildup and this contributes to lowering the ageof onset of AD, or the presence or absence of particular APOE allelesmay affect the way neurons respond to injury (Buttini et al., 1999).

Also Apo A1 has been shown to be amyloigenic. Intact apo A1 can itselfform amyloid-like fibrils in vitro that are Congo red positive (Am JPathol 147 (2): 238-244 (August 1995), Wisniewski T, Golabek A A, KidaE, Wisniewski K E, Frangione B).

There seem to be some contradictory results indicating that there is apositive effect of the APOE-ε4 allele in decreasing symptoms of mentalloss, compared to other alleles (Stern, Brandt, 1997, Annals ofNeurology 41).

Neurofibrillary Tangles

This second hallmark of AD consists of abnormal collections of twistedthreads found inside nerve cells. The chief component of tangles is oneform of a protein called tau (τ). In the central nervous system, tauproteins are best known for their ability to bind and help stabilizemicrotubules, which are one constituent of the cell's internal supportstructure, or skeleton. However, in AD tau is changed chemically, andthis altered tau can no longer stabilize the microtubules, causing themto fall disintegrate. This collapse of the transport system may at firstresult in malfunctions in communication between nerve cells and maylater lead to neuronal death.

In AD, chemically altered tau twists into paired helical filaments—twothreads of tau that are wound around each other. These filaments are themajor substance found in neurofibrillary tangles. In one recent study,researchers found neurofibrillary changes in fewer than 6 percent of theneurons in a particular part of the hippocampus in healthy brains, inmore than 43 percent of these neurons in people who died with mild AD,and in 71 percent of these neurons in people who died with severe AD.When the loss of neurons was studied, a similar progression was found.Evidence of this type supports the idea that the formation of tanglesand the loss of neurons progress together over the course of AD.(National Institute on Aging Progress Report on Alzheimer's Disease,1999).

Tauopathies and Tangles

Several neurodegenerative diseases, other than AD, are characterized bythe aggregation of tau into insoluble filaments in neurons and glia,leading to dysfunction and death. Very recently, several groups ofresearchers, who were studying families with a variety of hereditarydementias other than AD, found the first mutations in the tau gene onchromosome 17 (Clark et al., 1998; Hutton et al., 1998; Poorkaj et al.,1998; Spillantini et al., 1998). In these families, mutations in the taugene cause neuronal cell death and dementia. These disorders which sharesome characteristics with AD but differ in several important respects,are collectively called “fronto temporal dementia and parkinsonismlinked to chromosome 17” (FTDP-17). They are diseases such asParkinson's disease, some forms of amyotrophic lateral sclerosis (ALS),corticobasal degeneration, progressive supranuclear palsy, and Pick'sdisease, and are all characterized by abnormal aggregation of tauprotein.

Other AD-like Neurological Diseases.

There are important parallels between AD and other neurologicaldiseases, including prion diseases (such as kuru, Creutzfeld-Jacobdisease and bovine spongiform encephalitis), Parkinson's disease,Huntington's disease, and fronto-temporal dementia. All involve depositsof abnormal proteins in the brain. AD and prion diseases cause dementiaand death, and both are associated with the formation of insolubleamyloid fibrils, but from membrane proteins that are different from eachother.

Scientists studying Parkinson's disease, the second-most commonneurodegenerative disorder after AD, discovered the first gene linked tothe disease. This gene codes for a protein called synuclein, which,intriguingly, is also found in the amyloid plaques of AD patients'brains (Lavedan C, 1998, Genome Res. 8(9): 871-80). Investigators havealso discovered that genetic defects in Huntington's disease, anotherprogressive neurodegenerative disorder that causes dementia, cause theHuntington protein to form into insoluble fibrils very similar to the Aβfibrils of AD and the protein fibrils of prion disease, (Scherzinger E,et al., 1999, PNAS U.S.A. 96(8): 4604-9).

Scientists have also discovered a novel gene, which when mutated, isresponsible for familial British dementia (FBD), a rare inheriteddisease that causes severe movement disorders and progressive dementiasimilar to that seen in AD. In a biochemical analysis of the amyloidfibrils found in the FBD plaques, a unique peptide named ABri was found(Vidal et al., 1999). A mutation at a particular point along this generesults in the production of a longer-than-normal Bri protein. The ABripeptide, which is snipped from the mutated end of the Bri protein isdeposited as amyloid fibrils. These plaques are thought to lead to theneuronal dysfunction and dementia that characterizes FBD.

Immunization with Aβ

The immune system will normally take part in the clearing of foreignprotein and proteinaceous particles in the organism but the depositsassociated with the above-mentioned diseases consist mainly ofself-proteins, thereby rendering the role of the immune system in thecontrol of these diseases less obvious. Further, the deposits arelocated in a compartment (the CNS) normally separated from the immunesystem, both facts suggesting that any vaccine or immunotherapeuticalapproach would be unsuccessful.

Nevertheless, scientists have recently attempted immunizing mice with avaccine composed of heterologous human Aβ and a substance known toexcite the immune system (Schenk et al., 1999 and WO 99/27944). Thevaccine was tested in a partial transgenic mouse model of AD with ahuman mutated gene for APP inserted into the DNA of the mouse. The miceproduced the modified APP protein and developed amyloid plaques as theygrew older. This mouse model was used to test whether vaccinationagainst the modified transgenic human APP had an effect on plaquebuild-up. In a first experiment, one group of transgenic mice was givenmonthly injections of the vaccine starting at 6 weeks of age and endingat 11 months. A second group of transgenic mice received no injectionsand served as a control group. By 13 months of age, the mice in thecontrol group had plaques covering 2 to 6 percent of their brains. Incontrast, the immunized mice had virtually no plaques.

In a second experiment, the researchers began the injections at 11months, when some plaques had already developed. Over a 7-month period,the control transgenic mice had a 17-fold increase in the amount ofplaque in their brains, whereas those who received the vaccine had a99-percent decrease compared to the 18-month-old control transgenicmice. In some mice, some of the pre-existing plaque deposits appeared tohave been removed by the treatment. It was also found that otherplaque-associated damage, such as inflammation and abnormal nerve cellprocesses, lessened as a result of the immunization.

The above is thus a preliminary study in mice and for example,scientists need to find out whether vaccinated mice remain healthy inother respects and whether memory of those vaccinated remains normal.Furthermore, because the mouse model is not a complete representation ofAD (the animals do not develop neurofibrillary tangles nor do many oftheir neurons die), additional studies will be necessary to determinewhether humans have a similar or different reaction from mice. Anotherissue to consider is that the method may perhaps “cure” amyloiddeposition but fail to stop development of dementia.

Technical issues present major challenges as well. For example it isunlikely that it is even possible, using this technology, to create avaccine which enables humans to raise antibodies against their ownproteins. So numerous issues of safety and effectiveness will need to beresolved before any tests in humans can be considered.

The work by Schenk et al. thus shows that if it was possible to generatea strong immune response towards self-proteins in proteinaceous depositsin the central nervus system such as the plaques formed in AD, it ispossible to both prevent the formation of the deposits and possibly alsoclear already formed plaques.

Recently, clinical trials using the above-discussed Aβ vaccines havebeen terminated due to adverse effects: A number of the vaccinatedsubjects developed chronic encephalitis that may be due to anuncontrolled autoimmunity against Aβ in the CNS.

OBJECT OF THE INVENTION

The object of the present invention is to provide novel therapiesagainst conditions characterized by deposition of amyloid, such as AD. Afurther object is to develop an autovaccine against amyloid, in order toobtain a novel treatment for AD and for other pathological disordersinvolving amyloid deposition.

SUMMARY OF THE INVENTION

Described herein is the use of an autovaccination technology forgenerating strong immune responses against otherwise non-immunogenic APPand Aβ Described is also the preparation of such vaccines for theprevention, possible cure or alleviation of the symptoms of suchdiseases associated with amyloid deposits.

Thus, in its broadest and most general scope, the present inventionrelates to a method for in vivo down-regulation of amyloid precursorprotein (APP) or beta amyloid (Aβ) in an animal, including a humanbeing, the method comprising effecting presentation to the animal'simmune system of an immunogenically effective amount of at least oneanalogue of APP or Aβ that incorporates into the same molecule at leastone B-cell epitope of APP and/or Aβ and at least one foreign T-helperepitope (T_(H) epitope) so that immunization of the animal with theanalogue induces production of antibodies against the animal'sautologous APP or Aβ, wherein the analogue

-   -   a) is a polyamino acid that consists of at least one copy of a        subsequence of residues 672-714 in SEQ ID NO: 2, wherein the        foreign T_(H) epitope is incorporated by means of amino acid        addition and/or insertion and/or deletion and/or substitution,        wherein the subsequence is selected from the group consisting of        residues 1-42, residues 1-40, residues 1-39, residues 1-35,        residues 1-34, residues 1-28, residues 1-12, residues 1-5,        residues 13-28, residues 13-35, residues 17-28, residues 25-35,        residues 35-40, residues 36-42 and residues 35-42 of the amino        acid sequence consisting of amino acid residues 673-714 of SEQ        ID NO: 2; and/or    -   b) is a polyamino acid that contains the foreign T_(H) epitopes        and a disrupted APP or Aβ sequence so that the analogue does not        include any subsequence of SEQ ID NO: 2 that binds productively        to MHC class II molecules initiating a T-cell response; and/or    -   c) is a polyamino acid that comprises the foreign T_(H) epitope        and APP or Aβ derived amino acids, and comprises 1 single        methionine residue located in the C-terminus of the analogue,        wherein other methionine residues in APP or Aβ and in the        foreign T_(H) epitope have been substituted or deleted, and        preferably have been substituted by leucin or isoleucine; and/or    -   d) is a conjugate comprising a polyhydroxypolymer backbone to        which is separately coupled a polyamino acid as defined in a)        and/or a polyamino acid as defined in b) and/or a polyamino acid        as defined in c); and/or    -   e) is a conjugate comprising a polyhydroxypolymer backbone to        which is separately coupled 1) the foreign T_(H) epitope and 2)        a polyamino acid selected from the group consisting of a        subsequence as defined in a), a disrupted sequence of APP or Aβ        as defined in b), and an APP or Aβ derived amino acid sequence        that comprises 1 single methionine residue located in the        C-terminus, wherein other methionine residues in APP or Aβ and        in the foreign T_(H) epitope have been substituted or deleted,        and preferably have been substituted by leucin or isoleucine.

The present assignee has previously filed an international patentapplication directed to safe vaccination strategies againstamyloidogenic polypeptides such as APP and Aβ, cf. WO 01/62284. Thisapplication was not published on the filing date of the presentapplication and further does not contain details concerning theabove-mentioned useful analogues of APP and Aβ.

The invention also relates to analogues of the APP and Aβ as well as tonucleic acid fragments encoding a subset of these. Also immunogeniccompositions comprising the analogues or the nucleic acid fragments arepart of the invention.

LEGEND TO THE FIGURE

FIG. 1: Schematic depiction of Autovac variants derived from the amyloidprecursor protein with the purpose of generating antibody responsesagainst the Aβ protein Aβ-43 (or C-100). The APP is shown schematicallyat the top of the figure and the remaining schematic constructs showthat the model epitopes P2 and P30 are substituted or inserted intovarious truncations of APP. In the figure, the black pattern indicatesthe APP signal sequence, two-way cross-hatching is the extracellularpart of APP, dark vertical hatching is the transmembrane domain of APP,light vertical hatching is the intracellular domain of APP, coarsecross-hatching indicates the P30 epitope, and fine cross-hatchingindicates the P2 epitope. The full line box indicates Aβ-42/43 and thefull-line box and the dotted line box together indicate C-100. “Abeta”denotes Aβ.

FIG. 2: Schematic depiction of an embodiment of the synthesis ofgenerally applicable immunogenic conjugates. Peptide A (any antigenicsequence, e.g. an Aβ sequence described herein) and peptide B (an aminoacid sequence including a foreign T-helper epitope are synthesized andmixed. After that they are contacted with a suitable activatedpolyhydroxypolymer, peptides A and B are attached via the activationgroup in a ration corresponding to the initial ratio between these twosubstances in the peptide mixture. Cf. Example 4 for details.

DETAILED DISCLOSURE OF THE INVENTION DEFINITIONS

In the following a number of terms used in the present specification andclaims will be defined and explained in detail in order to clarify themetes and bounds of the invention.

The terms “amyloid” and “amyloid protein” which are used interchangeablyherein denote a class of proteinaceous unbranched fibrils ofindeterminate length. Amyloid fibrils display characteristic stainingwith Congo Red and share a cross-β structure in which the polypeptidechain is organized in β-sheets. Amyloid is generally derived fromamyloidogenic proteins which have very different precursor structuresbut which can all undergo a structural conversion to a misfolded formthat is the building block of the β-sheet helix protofilament. Normally,the diameter of amyloid fibrils varies between about 70 to about 120 Å.

The term “amyloidogenic protein” is intended to denote a polypeptidewhich is involved in the formation of amyloid deposits, either by beingpart of the deposits as such or by being part of the biosyntheticpathway leading to the formation of the deposits. Hence, examples ofamyloidogenic proteins are APP and Aβ, but also proteins involved in themetabolism of these may be amyloidogenic proteins.

An “amyloid polypeptide” is herein intended to denote polypeptidescomprising the amino acid sequence of the above-discussed amyloidogenicproteins derived from humans or other mammals (or truncates thereofsharing a substantial amount of B-cell epitopes with an intactamyloidogenic protein)—an amyloidogenic polypeptide can therefore e.g.comprise substantial parts of a precursor for the amyloidogenicpolypeptide (in the case of Aβ, one possible amyloid polypeptide couldbe APP derived). Also unglycosylated forms of amyloidogenic polypeptideswhich are prepared in prokaryotic system are included within theboundaries of the term as are forms having varying glycosylationpatterns due to the use of e.g. yeasts or other non-mammalian eukaryoticexpression systems. It should, however, be noted that when using theterm “an amyloidogenic polypeptide” it is intended that the polypeptidein question is normally non-immunogenic when presented to the animal tobe treated. In other words, the amyloidogenic polypeptide is aself-protein or is an analogue of such a self-protein which will notnormally give rise to an immune response against the amyloidogenic ofthe animal in question.

An “analogue” is an APP or Aβ derived molecule that incorporates one orseveral changes in its molecular structure. Such a change can e.g. be inthe form of fusion of APP or Aβ polyamino acids to a suitable fusionpartner (i.e. a change in primary structure exclusively involving C-and/or N-terminal additions of amino acid residues) and/or it can be inthe form of insertions and/or deletions and/or substitutions in thepolypeptide's amino acid sequence. Also encompassed by the term arederivatized APP or Aβ derived molecules, cf. the discussion below ofmodifications of APP or Aβ. In some cases the analogue may beconstructed so as to be less able or even unable to elicit antibodiesagainst the normal precursor protein(s) of the amyloid, thereby avoidingundesired interference with the (physiologically normal) non-aggregatedform of the polypeptide being a precursor of the amyloid protein.

It should be noted that the use as a vaccine in a human of axeno-analogue (e.g. a canine or porcine analogue) of a human APP or Aβcan be imagined to produce the desired immunity against the APP or Aβ.Such use of an xeno-analogue for immunization is also considered part ofthe invention.

The term “polypeptide” is in the present context intended to mean bothshort peptides of from 2 to 10 amino acid residues,, oligopeptides offrom 11 to 100 amino acid residues, and polypeptides of more than 100amino acid residues. Furthermore, the term is also intended to includeproteins, i.e. functional biomolecules comprising at least onepolypeptide; when comprising at least two polypeptides, these may formcomplexes, be covalently linked, or may be non-covalently linked. Thepolypeptide(s) in a protein can be glycosylated and/or lipidatedand/or-comprise prosthetic groups. Also, the term “polyamino acid” is anequivalent to the term “polypeptide”

The terms “T-lymphocyte” and “T-cell” will be used interchangeably forlymphocytes of thymic origin which are responsible for various cellmediated immune responses as well as for helper activity in the humoralimmune response. Likewise, the terms “B-lymphocyte” and “B-cell” will beused interchangeably for antibody-producing lymphocytes.

The term “subsequence” means any consecutive stretch of at least 3 aminoacids or, when relevant, of at least 3 nucleotides, derived directlyfrom a naturally occurring amyloid amino acid sequence or nucleic acidsequence, respectively.

The term “animal” is in the present context in general intended todenote an animal species (preferably mammalian), such as Homo sapiens,Canis domesticus, etc. and not just one single animal. However, the termalso denotes a population of such an animal species, since it isimportant that the individuals immunized according to the method of theinvention all harbour substantially the same APP or Aβ allowing forimmunization of the animals with the same immunogen(s). It will be clearto the skilled person that an animal in the present context is a livingbeing which has an immune system. It is preferred that the animal is avertebrate, such as a mammal.

By the term “in vivo down-regulation of APP or Aβ” is herein meantreduction in the living organism of the total amount of depositedamyloid protein (or amyloid as such) of the relevant type. Thedown-regulation can be obtained by means of several mechanisms: Ofthese, simple interference with amyloid by antibody binding so as toprevent misaggregation is the most simple. However, it is also withinthe scope of the present invention that the antibody binding results inremoval of amyloid by scavenger cells (such as macrophages and otherphagocytic cells) and that the antibodies interfer with otheramyloidogenic polypeptides which lead to amyloid formation. A furtherpossibility is that antibodies bind A outside the CNS, therebyeffectively removing Aβ from the CNS via a simple mass action principle.

The expression “effecting presentation . . . to the immune system” isintended to denote that the animal's immune system is subjected to animmunogenic challenge in a controlled manner. As will appear from thedisclosure below, such challenge of the immune system can be effected ina number of ways of which the most important are vaccination withpolypeptide containing “pharmaccines” (i.e. a vaccine which isadministered to treat or ameliorate ongoing disease) or nucleic acid“pharmaccine” vaccination. The important result to achieve is thatimmune competent cells in the animal are confronted with the antigen inan immunologically effective manner, whereas the precise mode ofachieving this result is of less importance to the inventive ideaunderlying the present invention.

The term “immunogenically effective amount” has its usual meaning in theart, i.e. an amount of an immunogen, which is capable of inducing animmune response that significantly engages pathogenic agents sharingimmunological features with the immunogen.

When using the expression that the APP or Aβ has been “modified” isherein meant that a chemical modification of the polypeptide has beenperformed on APP or Aβ. Such a modification can e.g. be derivatization(e.g. alkylation) of certain amino acid residues in the sequence, but aswill be appreciated from the disclosure below, the preferredmodifications comprise changes of the primary structure of the aminoacid sequence.

When discussing “autotolerance towards APP or Aβ” it is understood thatsince the polypeptide is a self-protein in the population to bevaccinated, normal individuals in the population do not mount an immuneresponse against the polypeptide; it cannot be excluded, though, thatoccasional individuals in an animal population might be able to produceantibodies against the native polypeptide, e.g. as part of an autoimmunedisorder. At any rate, an animal will normally only be autotoleranttowards its own APP or Aβ, but it cannot be excluded that analoguesderived from other animal species or from a population having adifferent phenotype would also be tolerated by said animal.

A “foreign T-cell epitope” (or: “foreign T-lymphocyte epitope”) is apeptide which is able to bind to an MHC molecule and which stimulatesT-cells in an animal species. Preferred foreign T-cell epitopes in theinvention are “promiscuous” epitopes, i.e. epitopes which bind to asubstantial fraction of a particular class of MHC molecules in an animalspecies or population. Only a very limited number of such promiscuousT-cell epitopes are known, and they will be discussed in detail below.Promiscuous T-cell epitopes are also denoted “universal” T-cellepitopes. It should be noted that in order for the immunogens which areused according to the present invention to be effective in as large afraction of an animal population as possible, it may be necessary to 1)insert several foreign T-cell epitopes in the same analogue or 2)prepare several analogues wherein each analogue has a differentpromiscuous epitope inserted. It should be noted also that the conceptof foreign T-cell epitopes also encompasses use of cryptic T-cellepitopes, i.e. epitopes which are derived from a self-protein and whichonly exerts immunogenic behaviour when existing in isolated form withoutbeing part of the self-protein in question.

A “foreign T helper lymphocyte epitope” (a foreign T_(H) epitope) is aforeign T cell epitope, which binds an MHC Class II molecule and can bepresented on the surface of an antigen presenting cell (APC) bound tothe MHC Class II molecule.

A “functional part” of a (bio)molecule is in the present contextintended to mean the part of the molecule which is responsible for atleast one of the biochemical or physiological effects exerted by themolecule. It is well-known in the art that many enzymes and othereffector molecules have an active site which is responsible for theeffects exerted by the molecule in question. Other parts of the moleculemay serve a stabilizing or solubility enhancing purpose and cantherefore be left out if these purposes are not of relevance in thecontext of a certain embodiment of the present invention. For instanceit is possible to use certain cytokines as a modifying moiety in APP orAβ (cf. the detailed discussion below), and in such a case, the issue ofstability may be irrelevant since the coupling to the APP or Aβmayprovide the stability necessary.

The term “adjuvant” has its usual meaning in the art of vaccinetechnology, i.e. a substance or a composition of matter which is 1) notin itself capable of mounting a specific immune response against theimmunogen of the vaccine, but which is 2) nevertheless capable ofenhancing the immune response against the immunogen. Or, in other words,vaccination with the adjuvant alone does not provide an immune responseagainst the immunogen, vaccination with the immunogen may or may notgive rise to an immune response against the immunogen, but the combinedvaccination with immunogen and adjuvant induces an immune responseagainst the immunogen which is stronger than that induced by theimmunogen alone.

“Targeting” of a molecule is in the present context intended to denotethe situation where a molecule upon introduction in the animal willappear preferentially in certain tissue(s) or will be preferentiallyassociated with certain cells or cell types. The effect can beaccomplished in a number of ways including formulation of the moleculein composition facilitating targeting or by introduction in the moleculeof groups, which facilitate targeting. These issues will be discussed indetail below.

“Stimulation of the immune system” means that a substance or compositionof matter exhibits a general, non-specific immunostimulatory effect. Anumber of adjuvants and putative adjuvants (such as certain cytokines)share the ability to stimulate the immune system. The result of using animmunostimulating agent is an increased “alertness” of the immune systemmeaning that simultaneous or subsequent immunization with an immunogeninduces a significantly more effective immune response compared toisolated use of the immunogen.

“Productive binding” means binding of a peptide to the MHC molecule(Class I or II) so as to be able to stimulate T-cells that engage a cellthat present the peptide bound to the MHC molecule. For instance, apeptide bound to an MHC Class II molecule on the surface of an APC issaid to be productively bound if this APC will stimulate a T_(H) cellthat binds to the presented peptide-MHC Class II complex.

Preferred Embodiments of Amyloid Down-regulation

It is preferred that the analogue used as an immunogen in the method ofthe invention is a modified APP or Aβ molecule wherein at least onechange is present in the amino acid sequence of the APP or Aβ, since thechances of obtaining the all-important breaking of autotolerance isgreatly facilitated that way—this is e.g. evident from the resultspresented in Example 2 herein, where immunization with wild-type Aβ iscompared to immunization with an Aβ variant molecule. It has been shown(in Dalum I et al., 1996, J. Immunol. 157: 4796-4804) that potentiallyself-reactive B-lymphocytes recognizing self-proteins arephysiologically present in normal individuals. However, in order forthese B-lymphocytes to be induced to actually produce antibodiesreactive with the relevant self-proteins, assistance is needed fromcytokine producing T-helper lymphocytes (T_(H)-cells orT_(H)-lymphocytes). Normally this help is not provided becauseT-lymphocytes in general do not recognize T-cell epitopes derived fromself-proteins when presented by antigen presenting cells (APCs).However, by providing an element of “foreignness” in a self-protein(i.e. by introducing an immunologically significant modification),T-cells recognizing the foreign element are activated upon recognizingthe foreign epitope on an APC (such as, initially, a mononuclear cell).Polyclonal B-lymphocytes (which are also APCs) capable of recognisingself-epitopes on the modified self-protein also internalise the antigenand subsequently presents the foreign T-cell epitope(s) thereof, and theactivated T-lymphocytes subsequently provide cytokine help to theseself-reactive polyclonal B-lymphocytes. Since the antibodies produced bythese polyclonal B-lymphocytes are reactive with different epitopes onthe modified polypeptide, including those which are also present in thenative polypeptide, an antibody cross-reactive with the non-modifiedself-protein is induced. In conclusion, the T-lymphocytes can be led toact as if the population of polyclonal B-lymphocytes have recognised anentirely foreign antigen, whereas in fact only the inserted epitope(s)is/are foreign to the host. In this way, antibodies capable ofcross-reacting with non-modified self-antigens are induced.

Several ways of modifying a peptide self-antigen in order to obtainbreaking of autotolerance are known in the art. It is neverthelesspreferred that the analogue according to the present invention includes

-   -   at least one first moiety is introduced which effects targeting        of the modified molecule to an antigen presenting cell (APC),        and/or    -   at least one second moiety is introduced which stimulates the        immune system, and/or    -   at least one third moiety is introduced which optimizes        presentation of the analogue to the immune system.

However, all these modifications should be carried out while maintaininga substantial fraction of the original B-lymphocyte epitopes in the APPor Aβ, since the B-lymphocyte recognition of the native molecule isthereby enhanced.

In one preferred embodiment, side groups (in the form of the foreign.T-cell epitopes or the above-mentioned first, second and third moieties)are covalently or non-covalently introduced. This is to mean thatstretches of amino acid residues derived from the APP or Aβ arederivatized without altering the primary amino acid sequence, or atleast without introducing changes in the peptide bonds between theindividual amino acids in the chain.

An alternative, and preferred, embodiment utilises amino acidsubstitution and/or deletion and/or insertion and/or addition (which maybe effected by recombinant means or by means of peptide synthesis;modifications which involves longer stretches of amino acids can giverise to fusion polypeptides). One especially preferred version of thisembodiment is the technique described in WO 95/05849, which discloses amethod for down-regulating self-proteins by immunising with analogues ofthe self-proteins wherein a number of amino acid sequence(s) has beensubstituted with a corresponding number of amino acid sequence(s) whicheach comprise a foreign immunodominant T-cell epitope, while at the sametime maintaining the overall tertiary structure of the self-protein inthe analogue. For the purposes of the present invention, it is howeversufficient if the modification (be it an insertion, addition, deletionor substitution) gives rise to a foreign T-cell epitope and at the sametime preserves a substantial number of the B-cell epitopes in the APP orAβ. However, in order to obtain maximum efficacy of the immune responseinduced, it is preferred that the overall tertiary structure of the APPor Aβ is maintained in the modified molecule.

In some cases, it is. preferred that the APP or Aβ or fragments thereofare mutated. Especially preferred are substitution variants where themethionine in position 35 in Aβ-43 has been substituted, preferably withleucine or isoleucine, or simply deleted. Especially preferred analoguescontain one single methionine that is located in the C-terminus, eitherbecause it is naturally occurring in the amyloidogenic polypeptide orforeign T_(H) epitope, or because it has been inserted or added. Hence,it also preferred that the part of the analogue that includes theforeign T_(H) epitope is free from methionine, except from the possibleC-terminal location of a methionine.

The main reason for removing all but one methionine is that it becomespossible to recombinantly prepare multimeric analogues that can besubsequently cleaved by cyanogenbromide to leave the single analogues.The advantage is, that recombinant production becomes facilitated thisway.

In fact, it is generally preferred that all analogues of APP or Aβ thatare used according to the present invention share the characteristic ofmerely including one single methionine that is positioned as theC-terminal amino acid in the analogue and that other methionines ineither the amyloidogenic polypeptide or the foreign T_(H) epitope aredeleted or substituted for another amino acid.

One further interesting mutation is a deletion or substitution of thephenylalanine in position 19 in Aβ-43, and it is especially preferredthat the mutation is a substitution of this phenylalanine residue with aproline.

Other interesting polyamino acids to be used in the analogues aretruncated parts of the Aβ-43 protein. These can also be employed inimmunogenic analogues according to the present invention. Especiallypreferred are the truncates Aβ(1-42), Aβ(1-40), Aβ(1-39), Aβ(1-35),Aβ(1-34), Aβ(1-34), Aβ(1-28), Aβ(1-12), Aβ(1-5), Aβ(13-28), Aβ(13-35),Aβ(17-28), Aβ(25-35), Aβ(35-40), Aβ(36-42), and Aβ(35-42) (where thenumbers in the parentheses indicate the amino acid stretches of Aβ-43that constitute the relevant fragment—Aβ(35-40) is e.g. identical toamino acids 706-711 in SEQ ID NO: 2). All these variants with truncatedparts of Aβ-43 can be made with the Aβ fragments described herein, inparticular with variants 9, 10, 11, 12, and 13 mentioned in Example 1.

The following formula describes the molecular constructs generallycovered by the invention:(MOD₁)_(s1)(amyloid_(e1))_(n1)(MOD₂)_(s2)(amyloide₂)_(n2) . . .(MOD_(x))_(sx)(amyloid_(ex))_(nx)   (I)where amyloid_(e1)-amyloid_(ex) are x B-cell epitope containingsubsequences of APP or Aβ which independently are identical ornon-identical and which may contain or not contain foreign side groups,x is an integer ≧3, n1-nx are x integers ≧0 (at least one is ≧1),MOD₁-MOD_(x) are x modifications introduced between the preserved B-cellepitopes, and s₁-s_(x) are x integers ≧0 (at least one is ≧1 if no sidegroups are introduced in the amyloid_(ex) sequences). Thus, given thegeneral functional restraints on the immunogenicity of the constructs,the invention allows for all kinds of permutations of the originalsequence of the APP or Aβ, and all kinds of modifications therein. Thus,included in the invention are modified APP or Aβ obtained by omission ofparts of the sequence which e.g. exhibit adverse effects in vivo oromission of parts which are normally intracellular and thus could giverise to undesired immunological reactions.

One preferred version of the constructs outlined above are, whenapplicable, those where the B-cell epitope containing subsequence of anamyloid protein is not extracellularly exposed in the precursorpolypeptide from which the amyloid is derived. By making such a choiceof the epitopes, it is ensured that antibodies are not generated whichwould be reactive with the cells producing the precursor and thereby theimmune response which is generated becomes limited to an immune responseagainst the undesired amyloid deposits. In this case it will e.g. befeasible to induce immunity against epitopes of APP or Aβ which are onlyexposed to the extracellular phase when being free from any coupling tothe cells from which they are produced.

Maintenance of a substantial fraction of B-cell epitopes or even theoverall tertiary structure of a protein which is subjected tomodification as described herein can be achieved in several ways. One issimply to prepare a polyclonal antiserum directed against thepolypeptide in question (e.g. an antiserum prepared in a rabbit) andthereafter use this antiserum as a test reagent (e.g. in a competitiveELISA) against the modified proteins which are produced. Modifiedversions (analogues) which react to the same extent with the antiserumas does the APP or Aβ must be regarded as having the same overalltertiary structure as APP or Aβ whereas analogues exhibiting a limited(but still significant and specific) reactivity with such an antiserumare regarded as having maintained a substantial fraction of the originalB-cell epitopes.

Alternatively, a selection of monoclonal antibodies reactive withdistinct epitopes on the APP or Aβ can be prepared and used as a testpanel. This approach has the advantage of allowing 1) an epitope mappingof the APP or Aβ and 2) a mapping of the epitopes which are maintainedin the analogues prepared.

Of course, a third approach would be to resolve the 3-dimensionalstructure of the APP or Aβ or of a biologically active truncate thereof(cf. above) and compare this to the resolved three-dimensional structureof the analogues prepared. Three-dimensional structure can be resolvedby the aid of X-ray diffraction studies and NMR-spectroscopy. Furtherinformation relating to the tertiary structure can to some extent beobtained from circular dichroism studies which have the advantage ofmerely requiring the polypeptide in pure form (whereas X-ray diffractionrequires the provision of crystallized polypeptide and NMR requires theprovision of isotopic variants of the polypeptide) in order to provideuseful information about the tertiary structure of a given molecule.However, ultimately X-ray diffraction and/or NMR are necessary to obtainconclusive data since circular dichroism can only provide indirectevidence of correct 3-dimensional structure via information of secondarystructure elements.

One preferred embodiment of the invention utilises multiplepresentations of B-lymphocyte epitopes of APP or Aβ (i.e. formula Iwherein at least one B-cell epitope is present in two positions). Thiseffect can be achieved in various ways, e.g. by simply preparing fusionpolypeptides comprising the structure (APP or Aβ derivedpolypeptide)_(m), where m is an integer ≧2 and then introduce themodfications discussed herein in at least one of the APP or Aβsequences. It is preferred that the modifications introduced includes atleast one duplication of a B-lymphocyte epitope and/or the introductionof a hapten. These embodiments including multiple presentations ofselected epitopes are especially preferred in situations where merelyminor parts of the APP or Aβ are useful as constituents in a vaccineagent.

As mentioned above, the introduction of a foreign T-cell epitope can beaccomplished by-introduction of at least one amino acid insertion,addition, deletion, or substitution. Of course, the normalsituation-will-be the introduction of more than one change in the aminoacid sequence (e.g. insertion of or substitution by a complete T-cellepitope) but the important goal to reach is that the analogue, whenprocessed by an antigen presenting cell (APC), will give rise to such aforeign immunodominant T-cell epitope being presented in context of anMCH Class II molecule on the surface of the APC. Thus, if the amino acidsequence of the APP or Aβ in appropriate positions comprises a number ofamino acid residues which can also be found in a foreign T_(H) epitopethen the introduction of a foreign T_(H) epitope can be accomplished byproviding the remaining amino acids of the foreign epitope by means ofamino acid insertion, addition, deletion and substitution. In otherwords, it is not necessary to introduce a complete T_(H) epitope byinsertion or substitution in order to fulfill the purpose of the presentinvention.

It is preferred that the number of amino acid insertions, deletions,substitutions or additions is at least 2, such as 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 25 insertions,substitutions, additions or deletions. It is furthermore preferred thatthe number of amino acid insertions, substitutions, additions ordeletions is not in excess of 150, such as at most 100, at most 90, atmost 80, and at most 70. It is especially preferred that the number ofsubstitutions, insertions, deletions, or additions does not exceed 60,and in particular the number should not exceed 50 or even 40. Mostpreferred is a number of not more than 30. With respect to amino acidadditions, it should be noted that these, when the resulting constructis in the form of a fusion polypeptide, is often considerably higherthan 150.

Preferred embodiments of the invention includes modification byintroducing at least one foreign immunodominant T-cell epitope. It willbe understood that the question of immune dominance of a T-cell epitopedepends on the animal species in question. As used herein, the term“immunodominance” simply refers to epitopes which in the vaccinatedindividual/population gives rise to a significant immune response, butit is a well-known fact that a T-cell epitope which is immunodominant inone individual/population is not necessarily immunodominant in anotherindividual of the same species, even though it may be capable of bindingMHC-II molecules in the latter individual. Hence, for the purposes ofthe present invention, an immune dominant T-cell epitope is a T-cellepitope which will be effective in providing T-cell help when present inan antigen. Typically, immune dominant T-cell epitopes has as aninherent feature that they will substantially always be presented boundto an MHC Class II molecule, irrespective of the polypeptide whereinthey appear. Another important point is the issue of MHC restriction ofT-cell epitopes. In general, naturally occurring T-cell epitopes are MHCrestricted, i.e. a certain peptides constituting a T-cell epitope willonly bind effectively to a subset of MHC Class II molecules. This inturn has the effect that in most cases the use of one specific T-cellepitope will result in a vaccine component which is only effective in afraction of the population, and depending on the size of that fraction,it can be necessary to include more T-cell epitopes in the samemolecule, or alternatively prepare a multi-component vaccine wherein thecomponents are variants of APP or Aβ which are distinguished from eachother by the nature of the T-cell epitope introduced.

If the MHC restriction of the T-cells used is completely unknown (forinstance in a situation where the vaccinated animal has a poorly definedMHC composition), the fraction of the population covered by a specificvaccine composition can be approximated by means of the followingformula $\begin{matrix}{f_{population} = {1 - {\prod\limits_{i = 1}^{n}\left( {1 - p_{i}} \right)}}} & ({II})\end{matrix}$where p_(i) is the frequency in the population of responders to the ithforeign T-cell epitope present in the vaccine composition, and n is thetotal number of foreign T-cell epitopes in the vaccine composition.Thus, a vaccine composition containing 3 foreign T-cell epitopes havingresponse frequencies in the population of 0.8, 0.7, and 0.6,respectively, would give1−0.2×0.3×0.4=0.976i.e. 97.6 percent of the population will statistically mount an MHC-IImediated response to the vaccine.

The above formula does not apply in situations where a more or lessprecise MHC restriction pattern of the peptides used is known. If, forinstance a certain peptide only binds the human MHC-II molecules encodedby HLA-DR alleles DR1, DR3, DR5, and DR7, then the use of this peptidetogether with another peptide which binds the remaining MHC-II moleculesencoded by HLA-DR alleles will accomplish 100% coverage in thepopulation in question. Likewise, if the second peptide only binds DR3and DR5, the addition of this peptide will not increase the coverage atall. If one bases the calculation of population response purely on MHCrestriction of T-cell epitopes in the vaccine, the minimum fraction ofthe population covered by a specific vaccine composition can bedetermined by means of the following formula: $\begin{matrix}{f_{population} = {1 - {\prod\limits_{j = 1}^{3}\left( {1 - \phi_{j}} \right)^{2}}}} & ({III})\end{matrix}$wherein φ_(j) is the sum of frequencies in the population of allelichaplotypes encoding MHC molecules which bind any one of the T-cellepitopes in the vaccine and which belong to the j^(th) of the 3 knownHLA loci (DP, DR and DQ); in practice, it is first determined which MHCmolecules will recognize each T-cell epitope in the vaccine andthereafter these are listed by type (DP, DR and DQ)—then, the individualfrequencies of the different listed allelic haplotypes are summed foreach type, thereby yielding φ₁, φ₂, and φ₃.

It may occur that the value p_(i) in formula II exceeds thecorresponding theoretical value π_(i): $\begin{matrix}{\pi_{i} = {1 - {\prod\limits_{j = 1}^{3}\left( {1 - v_{j}} \right)^{2}}}} & ({IV})\end{matrix}$wherein u_(j) is the sum of frequencies in the population of allelichaplotype encoding MHC molecules which bind the ith T-cell epitope inthe vaccine and which belong to the j^(th) of the 3 known HLA loci (DP,DR and DQ). This means that in 1−π_(i) of the population is a frequencyof responders of f_(residual) _(—) _(i)=(p_(i)−n_(i))/(1−n_(i)).Therefore, formula III can be adjusted so as to yield formula V:$\begin{matrix}{f_{population} = {1 - {\prod\limits_{j = 1}^{3}\left( {1 - \varphi_{j}} \right)^{2}} + \left( {1 - {\prod\limits_{i = 1}^{n}\left( {1 - f_{residual\_ i}} \right)}} \right)}} & (V)\end{matrix}$where the term 1−f_(residual-i) is set to zero if negative. It should benoted that formula V requires that all epitopes have been haplotypemapped against identical sets of haplotypes.

Therefore, when selecting T-cell epitopes to be introduced in theanalogue, it is important to include all knowledge of the epitopes whichis available: 1) The frequency of responders in the population to eachepitope, 2) MHC restriction data, and 3) frequency in the population ofthe relevant haplotypes.

There exist a number of naturally occurring “promiscuous” T-cellepitopes which are active in a large proportion of individuals of ananimal species or an animal population and these are preferablyintroduced in the vaccine thereby reducing the need for a very largenumber of different analogues in the same vaccine.

The promiscuous epitope can according to the invention be a naturallyoccurring human T-cell epitope such as epitopes from tetanus toxoid(e.g. the P2 and P30 epitopes), diphtheria toxoid, Influenza virushemagluttinin (HA), and P. falciparum CS antigen.

Over the years a number of other promiscuous T-cell epitopes have beenidentified. Especially peptides capable of binding a large proportion ofHLA-DR molecules encoded by the different HLA-DR alleles have beenidentified and these are all possible T-cell epitopes to be introducedin the analogues used according to the present invention. Cf. also theepitopes discussed in the following references which are hereby allincorporated by reference herein: WO 98/23635 (Frazer I H et al.,assigned to The University of Queensland); Southwood S et. al, 1998, J.Immunol. 160: 3363-3373; Sinigaglia F et al., 1988, Nature 336: 778-780;Chicz R M et al., 1993, J. Exp. Med 178: 27-47; Hammer J et al., 1993,Cell 74: 197-203; and Falk K et al., 1994, Immunogenetics 39: 230-242.The latter reference also deals with HLA-DQ and -DP ligands. Allepitopes listed in these 5 references are relevant as candidate naturalepitopes to be used in the present invention, as are epitopes whichshare common motifs with these.

Alternatively, the epitope can be any artificial T-cell epitope which iscapable of binding a large-proportion of MHC Class II molecules. In thiscontext the pan DR epitope peptides (“PADRE”) described in WO 95/07707and in the corresponding paper Alexander J et al., 1994, Immunity 1:751-761 (both disclosures are incorporated by reference herein) areinteresting candidates for epitopes to be used according to the presentinvention. It should be noted that the most effective PADRE peptidesdisclosed in these papers carry D-amino acids in the C- and N-termini inorder to improve stability when administered. However, the presentinvention primarily aims at incorporating the relevant epitopes as partof the analogue which should then subsequently be broken downenzymatically inside the lysosomal compartment of APCs to allowsubsequent presentation in the context of an MHC-II molecule andtherefore it is not expedient to incorporate D-amino acids in theepitopes used in the present invention.

One especially preferred PADRE peptide is the one having the amino acidsequence AKFVAAWTLKAAA (SEQ ID NO: 17) or an immunologically effectivesubsequence thereof. This, and other epitopes having the same lack ofMHC restriction are preferred T-cell epitopes which should be present inthe analogues used in the inventive method. Such super-promiscuousepitopes will allow for the most simple embodiments of the inventionwherein only one single analogue is presented to the vaccinated animal'simmune system.

As mentioned above, the modification of the APP or Aβ can also includethe introduction of a first moiety which targets the modifiedamyloidogenic polypeptide to an APC or a B-lymphocyte. For instance, thefirst moiety can be a specific binding partner for a B-lymphocytespecific surface antigen or for an APC specific surface antigen. Manysuch specific surface antigens are known in the art. For instance, themoiety can be a carbohydrate for which there is a receptor on theB-lymphocyte or the APC (e.g. mannan or mannose). Alternatively, thesecond moiety can be a hapten. Also an antibody fragment whichspecifically recognizes a surface molecule on APCs or lymphocytes can beused as a first moiety (the surface molecule can e.g. be an FCγ receptorof macrophages and monocytes, such as FCγRI or, alternatively any otherspecific surface marker such as CD40 or CTLA-4). It should be noted thatall these exemplary targeting molecules can be used as part of anadjuvant also, cf. below.

As an alternative or supplement to targeting the analogue to a certaincell type in order to achieve an enhanced immune response, it ispossible to increase the level of responsiveness of the immune system byincluding the above-mentioned second moiety which stimulates the immunesystem. Typical examples of such second moieties are cytokines, andheat-shock proteins or molecular chaperones, as well as effective partsthereof.

Suitable cytokines to be used according to the invention are those whichwill normally also function as adjuvants in a vaccine composition, i.e.for instance interferon γ (IFN-γ), interleukin 1 (IL-1), interleukin 2(IL-2), interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 12(IL-12), interleukin 13 (IL-13), interleukin 15 (IL-15), andgranulocyte-macrophage colony stimulating factor (GM-CSF);alternatively, the functional part of the cytokine molecule may sufficeas the second moiety. With respect to the use of such cytokines asadjuvant substances, cf. the discussion below.

According to the invention, suitable heat-shock proteins or molecularchaperones used as the second moiety can be HSP70, HSP90, HSC70, GRP94(also known as gp96, cf. Wearsch P A et al. 1998, Biochemistry 37:5709-19), and CRT (calreticulin).

Alternatively, the second moiety can be a toxin, such as listeriolycin(LLO), lipid A and heat-labile enterotoxin. Also, a number ofmycobacterial derivatives such as MDP (muramyl dipeptide), CFA (completeFreund's adjuvant) and the trehalose diesters TDM and TDE areinteresting possibilities.

Also the possibility of introducing a third moiety which enhances thepresentation of the analogue to the immune system is an importantembodiment of the invention. The art has shown several examples of thisprinciple. For instance, it is known that the palmitoyl lipidationanchor in the Borrelia burgdorferi protein OspA can be utilised so as toprovide self-adjuvating polypeptides (cf. e.g. WO 96/40718)—it seemsthat the lipidated proteins form up micelle-like structures with a coreconsisting of the lipidation anchor parts of the polypeptides and theremaining parts of the molecule protruding therefrom, resulting inmultiple presentations of the antigenic determinants. Hence, the use ofthis and related approaches using different lipidation anchors (e.g. amyristyl group, a myristyl group, a farnesyl group, a geranyl-geranylgroup, a GPI-anchor, and an N-acyl diglyceride group) are preferredembodiments of the invention, especially since the provision of such alipidation anchor in a recombinantly produced protein is fairlystraightforward and merely requires use of e.g. a naturally occurringsignal sequence as a fusion partner for the analogue. Anotherpossibility is use of the C3d fragment of complement factor C3 or C3itself (cf. Dempsey et al., 1996, Science 271, 348-350 and Lou & Kohler,1998, Nature Biotechnology 16, 458-462).

An alternative embodiment of the invention which also results in thepreferred presentation of multiple (e.g. at least 2) copies of theimportant epitopic regions of APP or Aβ to the immune system is thecovalent coupling of the analogue to certain molecules, i.e. variants dand e mentioned above. For instance, polymers can be used, e.g.carbohydrates such as dextran, cf. e.g. Lees A et al., 1994, Vaccine 12:1160-1166; Lees A et al., 1990, J Immunol. 145: 3594-3600, but alsomannose and mannan are useful alternatives. Integral membrane proteinsfrom e.g. E. coli and other bacteria are also useful conjugationpartners. The traditional carrier molecules such as keyhole limpethemocyanin (KLH), tetanus toxoid, diphtheria toxoid, and bovine serumalbumin (BSA) are also preferred and useful conjugation partners.

Preferred embodiments of covalent coupling of the APP or Aβ derivedmaterial to polyhydroxypolymers such as carbohydrates involve the use ofat least one APP or Aβ derived peptide and at least one foreign T-helperepitope which are coupled separately to the polyhydroxypolymer (i.e. theforeign T-helper epitope and the APP or Aβ derived amino acid sequenceare not fused to each other but rather bound to the polyhydroxypolymerwhich then serves as a carrier backbone). Again, such an embodiment ismost preferred when the suitable B-cell epitope carrying regions of theAPP or Aβ derived peptides are constituted by short peptidestretches—this is because this approach is one very convenient way toachieve multiple presentations of selected epitopes in the resultingimmunogenic agent. It is, however, also possible to simply coupledanalogues already described herein to the polyhydroxypolymer backbone,i.e. that the APP or Aβ derived material is not attached to the backboneseparately from the foreign T_(H) epitopes.

It is especially preferred that the coupling of the foreign T-helperepitope and the APP or Aβ derived (poly)peptide is by means of an amidebond which can be cleaved by a peptidase. This strategy has the effectthat APCs will be able to take up the conjugate and at the same time beable to process the conjugate and subsequently present the foreignT-cell epitope in an MHC Class II context.

One way of achieving coupling of peptides (both the APP or Aβ derivedpeptide of interest as well as the foreign epitope) is to activate asuitable polyhydroxypolymer with tresyl (trifluoroethylsulphonyl) groupsor other suitable activation groups such as maleimido, p-Nitrophenylcloroformate (for activation of OH groups and formation of a peptidebond between peptide and polyhydroxypolymer), and tosyl(p-toluenesulfonyl). It is e.g. possible to prepare activatedpolysaccharides as described in WO 00/05316 and U.S. Pat. No. 5,874,469(both incorporated by reference herein) and couple these to APP or Aβderived peptides or polyamino acids as well as to T-cell epitopesprepared by means of conventional solid or liquid phase peptidesynthesis techniques. The resulting product consists of apolyhydroxypolymer backbone (e.g. a dextran backbone) that has, attachedthereto by their N-termini or by other available nitrogen moieties,polyamino acids derived from APP or Ad and from foreign T-cell epitopes.If desired, it is possible to synthesise the APP or Aβ peptides so as toprotect all available amino groups but the one at the N-terminus,subsequently couple the resulting protected peptides to the tresylateddextran moiety, and finally de-protecting the resulting conjugate. Aspecific example of this approach is described in the examples below.

Instead of using the water-soluble polysaccharide molecules as taught inWO 00/05316 and U.S. Pat. No. 5,874,469, it is equally possible toutilise cross-linked polysaccharide molecules, thereby obtaining aparticulate conjugate between polypeptides and polysaccharide—this isbelieved to lead to an improved presentation to the immune system of thepolypeptides, since two goals are reached, namely to obtain a localdeposit effect when injecting the conjugate and to obtain particleswhich are attractive targets for APCs. The approach of using suchparticulate systems is also detailed in the examples.

Considerations underlying chosen areas of introducing modifications inAPP or Aβ are a) preservation of known and predicted B-cell epitopes, b)preservation of tertiary structure, c) avoidance of B-cell epitopespresent on “producer cells” etc. At any rate, as discussed above, it isfairly easy to screen a set of analogues which have all been subjectedto introduction of a T-cell epitope in different locations.

Since the most preferred embodiments of the present invention involvedown-regulation of human Aβ, it is consequently preferred that the APPor Aβpolypeptide discussed above is a human Aβ polypeptide. In thisembodiment, it is especially preferred that the APP or Aβ polypeptidehas been modified by substituting at least one amino acid sequence inSEQ ID NO: 2 with at least one amino acid sequence of equal or differentlength and containing a foreign T_(H) epitope. Preferred examples ofmodified amyloidogenic APP and Aβ are shown schematically in FIG. 1using the P2 and P30 epitopes as examples. The rationale behind suchconstructs is discussed in detail in the examples.

More specifically, a T_(H) containing (or completing) amino acidsequence which is introduced into SEQ ID NO: 2 may be introduced at anyamino acid in SEQ ID NO: 2. That is, the introduction is possible afterany of amino acids 1-770, but preferably after any of amino acids 671,672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685,686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699,700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713,and 714 in SEQ ID NO: 2. This may be combined with deletion of any orall of amino acids 1-671, or any of all of amino acids 715-770.Furthermore, when utilising the technique of substitution, any one ofamino acids 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682,683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696,697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710,711, 712, 713, and 714 in SEQ ID NO: 2 may be deleted in combinationwith the introduction.

Another embodiment of the present invention is the presentation of theanalogues which do not include any subsequence of of SEQ ID NO: 2 thatbinds productively to MHC class II molecules initiating a T-cellresponse.

The rationale behind such a strategy for design of the immunogen thatengages the immune system to induce e.g. an anti-Aβ immune response isthe following: It has been noted that when immunizing with abundantautologous proteins such as Aβ formulated in an adjuvant which issufficiently strong to break the body's tolerance towards the autologousprotein, there is a danger that in some vaccinated individuals theimmune response induced cannot be discontinued simply by discontinueingthe immunisation. This is because the induced immune response in suchindividuals is most likely driven by a native T_(H) epitope of theautologous protein, and this has the adverse effect that the vaccinatedindividual's own protein will be able to function as an immunizing agentin its own right: An autoimmune condition has thus been established.

The preferred methods including use of foreign T_(H) epitopes have tothe best of the inventors' knowledge never been observed to produce thiseffect, because the anti-self immune response is driven by a foreignT_(H) epitope, and it has been repeatedly demonstrated by the inventorsthat the induced immune response invoked by the preferred technologyindeed declines after discontinuation of immunizations. However, intheory it could happen in a few individuals that the immune responsewould also be driven by an autologous T_(H) epitope of the relevantself-protein one immunises against)—this is especially relevant whenconsidering self-proteins that are relatively abundant, such as Aβ,whereas other therapeutically relevant self-proteins are only presentlocally or in so low amounts in the body, that a “self-immunizationeffect” is not a possibility. One very simple way of avoiding this ishence to altogether avoid inclusion in the immunogen of peptidesequences that could serve as T_(H) epitopes (and since peptides shorterthan about 9 amino acids cannot serve as T_(H) epitopes, the use ofshorter fragments is one simple and feasible approach). Therefore, thisembodiment of the invention also serves to ensure that the immunogendoes not indlude peptide sequences of the target APP or Aβ that couldserve as “self-stimulating T_(H) epitopes” including sequences thatmerely contain conservative substitutions in a sequence of the targetprotein that might otherwise function as a T_(H) epitope.

Preferred embodiments of the immune system presentation of the analoguesof the APP or Aβ involve the use of a chimeric peptide comprising atleast one APP or Aβ derived peptide, which does not bind productively toMHC class II molecules, and at least one foreign T-helper epitope.Moreover, it is preferred that the APP or Aβ derived peptide harbours aB-cell epitope. It is especially advantageous if the immunogenicanalogue is one, wherein the amino acid sequences comprising one or moreB-cell epitopes are represented either as a continuous sequence or as asequence including inserts, wherein the inserts comprise foreignT-helper epitopes.

Again, such an embodiment is most preferred when the suitable B-cellepitope carrying regions of the APP or Aβ are constituted by shortpeptide stretches that in no way would be able to bind productively toan MHC Class II molecule. The selected B-cell epitope or -epitopes ofthe amyloidogenic polypeptide should therefore comprise at most 9consecutive amino acids of SEQ ID NO: 2. Shorter peptides are preferred,such as those having at most 8, 7, 6, 5, 4, or 3 consecutive amino acidsfrom the amyloidogenic polypeptide's amino acid sequence.

It is preferred that the analogue comprises at least one subsequence ofSEQ ID NO: 2 so that each such at least one subsequence independentlyconsists of amino acid stretches from the APP or Aβ selected from thegroup consisting of 9 consecutive amino acids, 8 consecutive aminoacids, 7 consecutive amino acids, 6 consecutive amino acids, 5consecutive amino acids, 4 consecutive amino acids, and 3 consecutiveamino acids.

It is especially preferred that the consecutive amino acids begins at anamino acid residue selected from the group consisting of residue 672,673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686,687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700,701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, and 714of SEQ ID NO: 2.

Protein/peptide Vaccination; Formulation and Administration of theAnalogues

When effecting presentation of the analogue to an animal's immune systemby means of administration thereof to the animal, the formulation of thepolypeptide follows the principles generally acknowledged in the art.

Preparation of vaccines which contain peptide sequences as activeingredients is generally well understood in the art, as exemplified byU.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792;and 4,578,770, all incorporated herein by reference. Typically, suchvaccines are prepared as injectables either as liquid solutions orsuspensions; solid forms suitable for solution in, or suspension in,liquid prior to injection may also be prepared. The preparation may alsobe emulsified. The active immunogenic ingredient is often mixed withexcipients which are pharmaceutically acceptable and compatible with theactive ingredient. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol, or the like, and combinations thereof. Inaddition, if desired, the vaccine may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,or adjuvants which enhance the effectiveness of the vaccines; cf. thedetailed discussion of adjuvants below.

The vaccines are conventionally administered parenterally, by injection,for example, either subcutaneously, intracutaneously, orintramuscularly. Additional formulations which are suitable for othermodes of administration include suppositories and, in some cases, oral,buccal, sublinqual, intraperitoneal, intravaginal, anal, epidural,spinal, and intracranial formulations. For suppositories, traditionalbinders and carriers may include, for example, polyalkalene glycols ortriglycerides; such suppositories may be formed from mixtures containingthe active ingredient in the range of 0.5% to 10%, preferably 1-2%. Oralformulations include such normally employed excipients as, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, and the like. Thesecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders and contain 10-95%of active ingredient, preferably 25-70%. For oral formulations, choleratoxin is an interesting formulation partner (and also a possibleconjugation partner).

The polypeptides may be formulated into the vaccine as neutral or saltforms. Pharmaceutically acceptable salts include acid addition salts(formed with the free amino groups of the peptide) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups may also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

The vaccines are administered in a manner compatible with the dosageformulation, and in such amount as will be therapeutically effective andimmunogenic. The quantity to be administered depends on the subject tobe treated, including, e.g., the capacity of the individual's immunesystem to mount an immune response, and the degree of protectiondesired. Suitable dosage ranges are of the order of several hundredmicrograms active ingredient per vaccination with a preferred range fromabout 0.1 μg to 2,000 μg (even though higher amounts in the 1-10 mgrange are contemplated), such as in the range from about 0.5 μg to 1,000μg, preferably in the range from 1 μg to 500 μg and especially in therange from about 10 μg to 100 μg. Suitable regimens for initialadministration and booster shots are also variable but are typified byan initial administration followed by subsequent inoculations or otheradministrations.

The manner of application may be varied widely. Any of the conventionalmethods for administration of a vaccine are applicable. These includeoral application on a solid physiologically acceptable base or in aphysiologically acceptable dispersion, parenterally, by injection or thelike. The dosage of the vaccine will depend on the route ofadministration and will vary according to the age of the person to bevaccinated and the formulation of the antigen.

Some of the polypeptides of the vaccine are sufficiently immunogenic ina vaccine, but for some of the others the immune response will beenhanced if the vaccine further comprises an adjuvant substance.

Various methods of achieving adjuvant effect for the vaccine are known.General principles and methods are detailed in “The Theory and PracticalApplication of Adjuvants”, 1995, Duncan E. S. Stewart-Tull (ed.), JohnWiley & Sons Ltd, ISBN 0-471-95170-6, and also in “Vaccines: NewGenerationn Immunological Adjuvants”, 1995, Gregoriadis G et al. (eds.),Plenum Press, New York, ISBN 0-306-45283-9, both of which are herebyincorporated by reference herein.

It is especially preferred to use an adjuvant which can be demonstratedto facilitate breaking of the autotolerance to autoantigens; in fact,this is essential in cases where unmodified amyloidogenic polypeptide isused as the active ingredient in the autovaccine. Non-limiting examplesof suitable adjuvants are selected from the group consisting of animmune targeting adjuvant; an immune modulating adjuvant such as atoxin, a cytokine, and a mycobacterial derivative; an oil formulation; apolymer; a micelle forming adjuvant; a saponin; an immunostimulatingcomplex matrix (ISCOM matrix); a particle; DDA; aluminium adjuvants; DNAadjuvants; γ-inulin; and an encapsulating adjuvant. In general it shouldbe noted that the disclosures above which relate to compounds and agentsuseful as first, second and third moieties in the analogues also refermutatis mutandis to their use in the adjuvant of a vaccine of theinvention.

The application of adjuvants include use of agents such as aluminumhydroxide or phosphate (alum), commonly used as 0.05 to 0.1 percentsolution in buffered saline, admixture with synthetic polymers of sugars(e.g. Carbopol®) used as 0.25 percent solution, aggregation of theprotein in the vaccine by heat treatment with temperatures rangingbetween 70° to 101° C. for 30 second to 2 minute periods respectivelyand also aggregation by means of cross-linking agents are possible.Aggregation by reactivation with pepsin treated antibodies (Fabfragments) to albumin, mixture with bacterial cells such as C. parvum orendotoxins or lipopolysaccharide components of gram-negative bacteria,emulsion in physiologically acceptable oil vehicles such as mannidemono-oleate (Aracel A) or emulsion with 20 percent solution of aperfluorocarbon (Fluosol-DA) used as a block substitute may also beemployed. Admixture with oils such as squalene and IFA is alsopreferred.

According to the invention DDA (dimethyldioctadecylammonium bromide) isan interesting candidate for an adjuvant as is DNA and γ-inulin, butalso Freund's complete and incomplete adjuvants as well as quillajasaponins such as QuilA and QS21 are interesting as is RIBI. Furtherpossibilities are monophosphoryl lipid A (MPL), the above mentioned C3and C3d, and muramyl dipeptide (MDP).

Liposome formulations are also known to confer adjuvant effects, andtherefore liposome adjuvants are preferred according to the invention.

Also immunostimulating complex matrix type (ISCOM® matrix) adjuvants arepreferred choices according to the invention, especially since it hasbeen shown that this type of adjuvants are capable of up-regulating MHCClass II expression by APCs. An ISCOM® matrix consists of (optionallyfractionated) saponins (triterpenoids) from Auillaja saponaria,cholesterol, and phospholipid. When admixed with the immunogenicprotein, the resulting particulate formulation is what is known as anISCOM particle where the saponin constitutes 60-70% w/w, the cholesteroland phospholipid 10-15% w/w, and the protein 10-15% w/w. Detailsrelating to composition and use of immunostimulating complexes can e.g.be found in the above-mentioned text-books dealing with adjuvants, butalso Morein B et al., 1995, Clin. Immunother. 3: 461-475 as well as BarrI G and Mitchell G F, 1996, Immunol. and Cell Biol. 74: 8-25 (bothincorporated by reference herein) provide useful instructions for thepreparation of complete immunostimulating complexes.

Another highly interesting (and thus, preferred) possibility ofachieving adjuvant effect is to employ the technique described inGosselin et al., 1992 (which is hereby incorporated by referenceherein). In brief, the presentation of a relevant antigen such as anantigen of the present invention can be enhanced by conjugating theantigen to antibodies (or antigen binding antibody fragments) againstthe Fcγ receptors on monocytes/macrophages. Especially conjugatesbetween antigen and anti-FcγRI have been demonstrated to enhanceimmunogenicity for the purposes of vaccination.

Other possibilities involve the use of the targeting and immunemodulating substances (i.a. cytokines) mentioned above as candidates forthe first and second moieties in the modified versions of amyloidogenicpolypeptides. In this connection, also synthetic inducers of cytokineslike poly I:C are possibilities.

Suitable mycobacterial derivatives are selected from the groupconsisting of muramyl dipeptide, complete Freund's adjuvant, RIBI, and adiester of trehalose such as TDM and TDE.

Suitable immune targeting adjuvants are selected from the groupconsisting of CD40 ligand and CD40 antibodies or specifically bindingfragments thereof (cf. the discussion above), mannose, a Fab fragment,and CTLA-4.

Suitable polymer adjuvants are selected from the group consisting of acarbohydrate such as dextran, PEG, starch, mannan, and mannose; aplastic polymer such as; and latex such as latex beads.

Yet another interesting way of modulating an immune response is toinclude the immunogen (optionally together with adjuvants andpharmaceutically acceptable carriers and vehicles) in a “virtual lymphnode” (VLN) (a proprietary medical device developed by ImmunoTherapy,Inc., 360 Lexington Avenue, New York, N.Y. 10017-6501). The VLN (a thintubular device) mimics the structrue and function of a lymph node.

Insertion of a VLN under the skin creates a site of sterile inflammationwith an upsurge of cytokines and chemokines. T- and B-cells as well asAPCs rapidly respond to the danger signals, home to the inflamed siteand accumulate inside the porous matrix of the VLN. It has been shownthat the necessary antigen dose required to mount an immune response toan antigen is reduced when using the VLN and that immune protectionconferred by vaccination using a VLN surpassed conventional immunizationusing Ribi as an adjuvant. The technology is i.a. described briefly inGelber C et al., 1998, “Elicitation of Robust Cellular and HumoralImmune Responses to Small Amounts of Immunogens Using a Novel MedicalDevice Designated the Virtual Lymph Node”, in: “From the Laboratory tothe Clinic, Book of Abstracts, Oct. 12^(th)-15^(th) 1998, SeascapeResort, Aptos, Calif.”.

Microparticle formulation of vaccines has been shown in many cases toincrease the immunogenicity of protein antigens and is therefore anotherpreferred embodiment of the invention. Microparticles are made either asco-formulations of antigen with a polymer, a lipid, a carbohydrate orother molecules suitable for making the particles, or the microparticlescan be homogeneous particles consisting of only the antigen itself.

Examples of polymer based microparticles are PLGA and PVP basedparticles (Gupta, R. K. et. al. 1998) where the polymer and the antigenare condensed into a solid particle. Lipid based particles can be madeas micelles of the lipid (so-called liposomes) entrapping the antigenwithin the micelle (Pietrobon, P. J. 1995). Carbohydrate based particlesare typically made of a suitable degradable carbohydrate such as starchor chitosan. The carbohydrate and the antigen are mixed and condensedinto particles in a process similar to the one used for polymerparticles (Kas, H. S. et. al. 1997).

Particles consisting only of the antigen can be made by various sprayingand freeze-drying techniques. Especially suited for the purporses of thepresent invention is the super critical fluid technology that is used tomake very uniform particles of controlled size (York, P. 1999 &Shekunov, B. et. al. 1999).

It is expected that the vaccine should be administered 1-6 times peryear, such as 1, 2, 3, 4, 5, or 6 times a year to an individual in needthereof. It has previously been shown that the memory immunity inducedby the use of the preferred autovaccines according to the invention isnot permanent, and therefore the immune system needs to be periodicallychallenged with the amyloidogenic polypeptide or modified amyloidogenicpolypeptides.

Due to genetic variation, different individuals may react with immuneresponses of varying strength to the same polypeptide. Therefore, thevaccine according to the invention may comprise several differentpolypeptides in order to increase the immune response, cf. also thediscussion above concerning the choice of foreign T-cell epitopeintroductions. The vaccine may comprise two or more polypeptides, whereall of the polypeptides are as defined above.

The vaccine may consequently comprise 3-20 different modified orunmodified polypeptides, such as 3-10 different polypeptides.

Nucleic Acid Vaccination

As an alternative to classic administration of a peptide-based vaccine,the technology of nucleic acid vaccination (also known as “nucleic acidimmunisation”, “genetic immunisation”, and “gene immunisation”) offers anumber of attractive features.

First, in contrast to the traditional vaccine approach, nucleic acidvaccination does not require resource consuming large-scale productionof the immunogenic agent (e.g. in the form of industrial scalefermentation of microorganisms producing modified amyloidogenicpolypeptides). Furthermore, there is no need to device purification andrefolding schemes for the immunogen. And finally, since nucleic acidvaccination relies on the biochemical apparatus of the vaccinatedindividual in order to produce the expression product of the nucleicacid introduced, the optimum post-translational processing of theexpression product is expected to occur; this is especially important inthe case of autovaccination, since, as mentioned above, a significantfraction of the original B-cell epitopes should be preserved in themodified molecule, and since B-cell epitopes in principle can beconstituted by parts of any (bio)molecule (e.g. carbohydrate, lipid,protein etc.). Therefore, native glycosylation and lipidation patternsof the immunogen may very well be of importance for the overallimmunogenicity and this is best ensured by having the host producing theimmunogen.

Hence, a preferred embodiment of the invention's variants a-c compriseseffecting presentation of the analogue to the immune system byintroducing nucleic acid(s) encoding the analogue into the animal'scells and thereby obtaining in vivo expression by the cells of thenucleic acid(s) introduced.

In this embodiment, the introduced nucleic acid is preferably DNA whichcan be in the form of naked DNA, DNA formulated with charged oruncharged lipids, DNA formulated in liposomes, DNA included in a viralvector, DNA formulated with a transfection-facilitating protein orpolypeptide, DNA formulated with a targeting protein or polypeptide, DNAformulated with Calcium precipitating agents, DNA coupled to an inertcarrier molecule, DNA encapsulated in a polymer, e.g. in PLGA (cf. themicroencapsulation technology described in WO 98/31398) or in chitin orchitosan, and DNA formulated with an adjuvant. In this context it isnoted that practically all considerations pertaining to the use ofadjuvants in traditional vaccine formulation apply for the formulationof DNA vaccines. Hence, all disclosures herein which relate to use ofadjuvants in the context of polypeptide based vaccines apply mutatismutandis to their use in nucleic acid vaccination technology.

As for routes of administration and administration schemes ofpolypeptide based vaccines which have been detailed above, these arealso applicable for the nucleic acid vaccines of the invention and alldiscussions above pertaining to routes of administration andadministration schemes for polypeptides apply mutatis mutandis tonucleic acids. To this should be added that nucleic acid vaccines cansuitably be administered intraveneously and intraarterially.Furthermore, it is well-known in the art that nucleic acid vaccines canbe administered by use of a so-called gene gun, and hence also this andequivalent modes of administration are regarded as part of the presentinvention. Finally, also the use of a VLN in the administration ofnucleic acids has been reported to yield good results, and thereforethis particular mode of administration is particularly preferred.

Furthermore, the nucleic acid(s) used as an immunization agent cancontain regions encoding the 1^(st), 2^(nd) and/or 3^(rd) moieties, e.g.in the form of the immunomodulating substances described above such asthe cytokines discussed as useful adjuvants. A preferred version of thisembodiment encompasses having the coding region for the analogue and thecoding region for the immunomodulator in different reading frames or atleast under the control of different promoters. Thereby it is avoidedthat the analogue or epitope is produced as a fusion partner to theimmunomodulator. Alternatively, two distinct nucleotide fragments can beused, but this is less preferred because of the advantage of ensuredco-expression when having both coding regions included in the samemolecule.

Accordingly, the invention also relates to a composition for inducingproduction of antibodies against APP or Aβ, the composition comprising

-   -   a nucleic acid fragment or a vector of the invention (cf. the        discussion of vectors below), and    -   a pharmaceutically and immunologically acceptable vehicle and/or        carrier and/or adjuvant as discussed above.

Under normal circumstances, the variant-encoding nucleic acid isintroduced in the form of a vector wherein expression is under controlof a viral promoter. For more detailed discussions of vectors accordingto the invention, cf. the discussion below. Also, detailed disclosuresrelating to the formulation and use of nucleic acid vaccines areavailable, cf. Donnelly J J et al, 1997, Annu. Rev. Immunol. 15: 617-648and Donnelly J J et al., 1997, Life Sciences 60: 163-172. Both of thesereferences are incorporated by reference herein.

Live Vaccines

A third alternative for effecting presentation of the analogues as theseare defined in variants a-c to the immune system is the use of livevaccine technology. In live vaccination, presentation to the immunesystem is effected by administering, to the animal, a non-pathogenicmicroorganism which has been transformed with a nucleic acid fragmentencoding an analogue or with a vector incorporating such a nucleic acidfragment. The non-pathogenic microorganism can be any suitableattenuated bacterial strain (attenuated by means of passaging or bymeans of removal of pathogenic expression products by recombinant DNAtechnology), e.g. Mycobacterium bovis BCG., non-pathogenic Streptococcusspp., E. coli, Salmonella spp., Vibrio cholerae, Shigella, etc. Reviewsdealing with preparation of state-of-the-art live vaccines can e.g. befound in Saliou P, 1995, Rev. Prat. 45: 1492-1496 and Walker P D, 1992,Vaccine 10: 977-990, both incorporated by reference herein. For detailsabout the nucleic acid fragments and vectors used in such live vaccines,cf. the discussion below.

As an alternative to bacterial live vaccines, the nucleic acid fragmentof the invention discussed below can be incorporated in a non-virulentviral vaccine vector such as a vaccinia strain or any other suitablepoxvirus.

Normally, the non-pathogenic microorganism or virus is administered onlyonce to the animal, but in certain cases it may be necessary toadminister the microorganism more than once in a lifetime in order tomaintain protective immunity. It is even contemplated that immunizationschemes as those detailed above for polypeptide vaccination will beuseful when using live or virus vaccines.

Alternatively, live or virus vaccination is combined with previous orsubsequent polypeptide and/or nucleic acid vaccination. For instance, itis possible to effect primary immunization with a live or virus vaccinefollowed by subsequent booster immunizations using the polypeptide ornucleic acid approach.

The microorganism or virus can be transformed with nucleic acid(s)containing regions encoding the 1^(st), 2^(nd) and/or 3^(rd) moieties,e.g. in the form of the immunomodulating substances described above suchas the cytokines discussed as useful adjuvants. A preferred version ofthis embodiment encompasses having the coding region for the analogueand the coding region for the immunomodulator in different readingframes or at least under the control of different promoters. Thereby itis avoided that the analogue or epitopes are produced as fusion partnersto the immunomodulator. Alternatively, two distinct nucleotide fragmentscan be used as transforming agents. Of course, having the 1^(st) and/or2^(nd) and/or 3^(rd) moieties in the same reading frame can provide asan expression product, an analogue of the invention, and such anembodiment is especially preferred according to the present invention.

Use of the Method of the Invention in Disease Treatment

As will be appreciated from the discussions above, the provision of themethod of the invention allows for control of diseases characterized byamyloid deposits. In this context, AD is the key target for theinventive method but also other diseases characterized by Aβ containingamyloid deposits are feasible targets. Hence, an important embodiment ofthe method of the invention for down-regulating amyloid activitycomprises treating and/or preventing and/or ameliorating AD or otherdiseases characterized by amyloid deposition, the method comprisingdown-regulating APP or Aβ according to the method of the invention tosuch an extent that the amount of amyloid is significantly decreased.

It is especially preferred that the reduction in amyloid results in aninversion of the balance between amyloid formation and amyloiddegradation/removal, i.e. that the rate of amyloid degradation/removalis brought to exceed the rate of amyloid formation. By carefullycontrolling the number and immunological impact of immunizations of theindividual in need thereof it will be possible to obtain a balance overtime which results in a net reduction of amyloid deposits without havingexcessive adverse effects.

Alternatively, if in an individual the method of the invention cannotremove or reduce existing amyloid deposits, the method of the inventioncan be used to obtain a clinically significant reduction in theformation of new amyloid, thereby significantly prolonging the timewhere the disease condition is non-debilitating. It should be possibleto monitor the rate of amyloid depositing by either measuring the serumconcentration of amyloid (which is believed to be in equilibrium withthe deposited material), or by using positron-emission tomography (PET)scanning, cf. Small G W, et al., 1996, Ann N Y Acad Sci 802: 70-78.

Other diseases and conditions where the present means and methods may beused in treatment or amelioration in an analogous way have beenmentioned above in the “Background of the invention” or are listed belowin the section headed “other amyloidic diseases and proteins associatedtherewith”.

Peptides, Polypeptides, and Compositions of the Invention

As will be apparent from the above, the present invention is based onthe concept of immunising individuals against the APP or Aβ antigen inorder to obtain a reduced amount of pathology-related amyloid deposits.The preferred way of obtaining such an immunization is to use theanalogues described herein, thereby providing molecules which have notpreviously been disclosed in the art.

It is believed that the analogues discussed herein are inventive intheir own right, and therefore an important part of the inventionpertains to an analogue as described above. Hence, any disclosurepresented herein pertaining to modified APP or Aβ are relevant for thepurpose of describing the amyloidogenic analogues of the invention, andany such disclosures apply mutatis mutandis to the description of theseanalogues.

It should be noted that preferred modified APP or Aβ molecules comprisemodifications which results in a polypeptide having a sequence identityof at least 70% with APP or Aβ or with a subsequence thereof of at least10 amino acids in length. Higher sequence identities are preferred, e.g.at least 75% or even at least 80, 85, 90, or 95%. The sequence identityfor proteins and nucleic acids can be calculated as(N_(ref)−N_(dif))·100/N_(ref), wherein N_(dif) is the total number ofnon-identical residues in the two sequences when aligned and whereinN_(ref) is the number of residues in one of the sequences. Hence, theDNA sequence AGTCAGTC will have a sequence identity of 75% with thesequence AATCAATC (N_(dif)=2 and N_(ref)=8).

The invention also pertains to compositions useful in exercising themethod of the invention. Hence, the invention also relates to animmunogenic composition comprising an immunogenically effective amountof an analouge as described above, said composition further comprising apharmaceutically and immunologically acceptable diluent and/or vehicleand/or carrier and/or excipient and optionally an adjuvant. In otherwords, this part of the invention concerns formulations of analogues,essentially as described above. The choice of adjuvants, carriers, andvehicles is accordingly in line with what has been discussed above whenreferring to formulation of modified and unmodified amyloidogenicpolypeptide for use in the inventive method for the down-regulation ofAPP or Aβ.

The polypeptides are prepared according to methods well-known in theart. Longer polypeptides are normally prepared by means of recombinantgene technology including introduction of a nucleic acid sequenceencoding the analogue into a suitable vector, transformation of asuitable host cell with the vector, expression by the host cell of thenucleic acid sequence, recovery of the expression product from the hostcells or their culture supernatant, and subseqeunt purification andoptional further modification, e.g. refolding or derivatization.

Shorter peptides are preferably prepared by means of the well-knowntechniques of solid- or liquid-phase peptide synthesis. However, recentadvances in this technology has rendered possible the production offull-length polypeptides and proteins by these means, and therefore itis also within the scope of the present invention to prepare the longconstructs by synthetic means.

Nucleic Acid Fragments and Vectors of the Invention

It will be appreciated from the above disclosure that polyamino acidanalogues can be prepared by means of recombinant gene technology butalso by means of chemical synthesis or semisynthesis; the latter twooptions are especially relevant when the modification consists incoupling to protein carriers (such as KLH, diphtheria toxoid, tetanustoxoid, and BSA) and non-proteinaceous molecules such as carbohydratepolymers and of course also when the modification comprises addition ofside chains or side groups to an APP or Aβ derived peptide chain.

For the purpose of recombinant gene technology, and of course also forthe purpose of nucleic acid immunization, nucleic acid fragmentsencoding analogues are important chemical products. Hence, an importantpart of the invention pertains to a nucleic acid fragment which encodesan analogue of the invention, i.e. an APP or Aβ derived polypeptidewhich either comprises the natural sequence to which has been added orinserted a fusion partner or, preferably an APP or Aβ derivedpolypeptide wherein has been introduced a foreign T-cell epitope bymeans of insertion and/or addition, preferably by means of substitutionand/or deletion. The nucleic acid fragments of the invention are eitherDNA or RNA fragments.

The nucleic acid fragments of the invention will normally be inserted insuitable vectors to form cloning or expression vectors carrying thenucleic acid fragments of the invention; such novel vectors are alsopart of the invention. Details concerning the construction of thesevectors of the invention will be discussed in context of transformedcells and microorganisms below. The vectors can, depending on purposeand type of application, be in the form of plasmids, phages, cosmids,mini-chromosomes, or virus, but also naked DNA which is only expressedtransiently in certain cells is an important vector. Preferred cloningand expression vectors of the invention are capable of autonomousreplication, thereby enabling high copy-numbers for the purposes ofhigh-level expression or high-level replication for subsequent cloning.

The general outline of a vector of the invention comprises the followingfeatures in the 5′-3′ direction and in operable linkage: a promoter fordriving expression of the nucleic acid fragment of the invention,optionally a nucleic acid sequence encoding a leader peptide enablingsecretion (to the extracellular phase or, where applicable, into theperiplasma) of or integration into the membrane of the polypeptidefragment, the nucleic acid fragment of the invention, and optionally anucleic acid sequence encoding a terminator. When operating withexpression vectors in producer strains or cell-lines it is for thepurposes of genetic stability of the transformed cell preferred that thevector when introduced into a host cell is integrated in the host cellgenome. In contrast, when working with vectors to be used for effectingin vivo expression in an animal (i.e. when using the vector in DNAvaccination) it is for security reasons preferred that the vector isincapable of being integrated in the host cell genome; typically, nakedDNA or non-integrating viral vectors are used, the choices of which arewell-known to the person skilled in the art

The vectors of the invention are used to transform host cells to producethe analogue of the invention. Such transformed cells, which are alsopart of the invention, can be cultured cells or cell lines used forpropagation of the nucleic acid fragments and vectors of the invention,or used for recombinant production of the analogues of the invention.Alternatively, the transformed cells can be suitable live vaccinestrains wherein the nucleic acid fragment (one single or multiplecopies) have been inserted so as to effect secretion or integration intothe bacterial membrane or cell-wall of the analogue.

Preferred transformed cells of the invention are microorganisms such asbacteria (such as the species Escherichia [e.g. E.coli], Bacillus [e.g.Bacillus subtilis], Salmonella, or Mycobacterium [preferablynon-pathogenic, e.g. M. bovis BCG]), yeasts (such as Saccharomycescerevisiae), and protozoans. Alternatively, the transformed cells arederived from a multicellular organism such as a fungus, an insect cell,a plant cell, or a mammalian cell. Most preferred are cells derived froma human being, cf. the discussion of cell lines and vectors below.Recent results have shown great promise in the use of a commerciallyavailable Drosophila melanogaster cell line (the Schneider 2 (S₂) cellline and vector system available from Invitrogen) for the recombinantproduction of polypeptides in applicants' lab, and therefore thisexpression system is particularly preferred.

For the purposes of cloning and/or optimized expression it is preferredthat the transformed cell is capable of replicating the nucleic acidfragment of the invention. Cells expressing the nucleic fragment arepreferred useful embodiments of the invention; they can be used forsmall-scale or large-scale preparation of the analogue of the inventionor, in the case of non-pathogenic bacteria, as vaccine constituents in alive vaccine.

When producing the analogues of the invention by means of transformedcells, it is convenient, although far from essential, that theexpression product is either exported out into the culture medium orcarried on the surface of the transformed cell.

When an effective producer cell has been identified it is preferred, onthe basis thereof, to establish a stable cell line which carries thevector of the invention and which expresses the nucleic acid fragmentencoding the modified amyloidogenic polypeptide. Preferably, this stablecell line secretes or carries the analogue of the invention, therebyfacilitating purification thereof.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with the hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using pBR322, a plasmid derived from an E. colispecies (see, e.g., Bolivar et al., 1977). The pBR322 plasmid containsgenes for ampicillin and tetracycline resistance and thus provides easymeans for identifying transformed cells. The pBR plasmid, or othermicrobial plasmid or phage must also contain, or be modified to contain,promoters which can be used by the prokaryotic microorganism forexpression.

Those promoters most commonly used in recombinant DNA constructioninclude the B-lactamase (penicillinase) and lactose promoter systems(Chang et al., 1978; Itakura et al., 1977; Goeddel et al., 1979) and atryptophan (trp) promoter system (Goeddel et al., 1979; EP-A-0 036 776).While these are the most commonly used, other microbial promoters havebeen discovered and utilized, and details concerning their nucleotidesequences have been published, enabling a skilled worker to ligate themfunctionally with plasmid vectors (Siebwenlist et al., 1980). Certaingenes from prokaryotes may be expressed efficiently in E. coli fromtheir own promoter sequences, precluding the need for addition ofanother promoter by artificial means.

In addition to prokaryotes, eukaryotic microbes, such as yeast culturesmay also be used, and here the promoter should be capable of drivingexpression. Saccharomiyces cerevisiase, or common baker's yeast is themost commonly used among eukaryotic microorganisms, although a number ofother strains are commonly available. For expression in Saccharomyces,the plasmid YRp7, for example, is commonly used (Stinchcomb et al.,1979; Kingsman et al., 1979; Tschemper et al., 1980). This plasmidalready contains the trpl gene which provides a selection marker for amutant strain of yeast lacking the ability to grow in tryptophan forexample ATCC No. 44076 or PEP4-1 (Jones, 1977). The presence of the trpllesion as a characteristic of the yeast host cell genome then providesan effective environment for detecting transformation by growth in theabsence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters for3-phosphoglycerate kinase (Hitzman et al., 1980) or other glycolyticenzymes (Hess et al., 1968; Holland et al., 1978), such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. In constructing suitableexpression plasmids, the termination sequences associated with thesegenes are also ligated into the expression vector 3′ of the sequencedesired to be expressed to provide polyadenylation of the mRNA andtermination.

Other promoters, which have the additional advantage of transcriptioncontrolled by growth conditions are the promoter region for alcoholdehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymesassociated with nitrogen metabolism, and the aforementionedglyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible formaltose and galactose utilization. Any plasmid vector containing ayeast-compatible promoter, origin of replication and terminationsequences is suitable.

In addition to microorganisms, cultures of cells derived frommulticellular organisms may also be used as hosts. In principle, anysuch cell culture is workable, whether from vertebrate or invertebrateculture. However, interest has been greatest in vertebrate cells, andpropagation of vertebrate in culture (tissue culture) has become aroutine procedure in recent years (Tissue Culture, 1973). Examples ofsuch useful host cell lines are VERO and HeLa cells, Chinese hamsterovary (CHO) cell lines, and W138, BHK, COS-7 293, Spodoptera frugiperda(SF) cells (commercially available as complete expression systems fromi.a. Protein Sciences, 1000 Research Parkway, Meriden, Conn. 06450,U.S.A. and from Invitrogen), and MDCK cell lines. In the presentinvention, an especially preferred cell line is S₂ available fromInvitrogen, PO Box 2312, 9704 CH Groningen, The Netherlands.

Expression vectors for such cells ordinarily include (if necessary) anorigin of replication, a promoter located in front of the gene to beexpressed, along with any necessary ribosome binding sites, RNA splicesites, polyadenylation site, and transcriptional terminator sequences.

For use in mammalian cells, the control functions on the expressionvectors are often provided by viral material. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, and most frequentlySimian Virus 40 (SV40). The early and late promoters of SV40 virus areparticularly useful because both are obtained easily from the virus as afragment which also contains the SV40 viral origin of replication (Fierset al., 1978). Smaller or larger SV40 fragments may also be used,provided there is included the approximately 250 bp sequence extendingfrom the HindIII site toward the BglI site located in the viral originof replication. Further, it is also possible, and often desirable, toutilize promoter or control sequences normally associated with thedesired gene sequence, provided such control sequences are compatiblewith the host cell systems.

An origin of replication may be provided either by construction of thevector to include an exogenous origin, such as may be derived from SV40or other viral (e.g., Polyoma, Adeno, VSV, BPV) or may be provided bythe host cell chromosomal replication mechanism. If the vector isintegrated into the host cell chromosome, the latter is oftensufficient.

Identification of Useful Analogues

It will be clear to the skilled person that not all possible variants ormodifications of naturally occurring APP or Aβ will have the ability toelicit antibodies in an animal which are cross-reactive with the naturalform. It is, however, not difficult to set up an effective standardscreen for modified amyloidogenic molecules which fulfill the minimumrequirements for immunological reactivity discussed herein. Hence, it ispossible to utilise a method for the identification of a modifiedamyloidogenic polypeptide which is capable of inducing antibodiesagainst unmodified amyloidogenic polypeptide in an animal species wherethe unmodified amyloidogenic polypeptide is a (non-immunogenic)self-protein, the method comprising

-   -   preparing, by means of peptide synthesis or genetic engineering        techniques, a set of mutually distinct analogue of the invention        wherein amino acids have been added to, inserted in, deleted        from, or substituted into the amino acid sequence of an APP or        Aβ of the animal species thereby giving rise to amino acid        sequences in the set which comprise T-cell epitopes which are        foreign to the animal species, or preparing a set of nucleic        acid fragments encoding the set of mutually distinct analogues,    -   testing members of the set of analogues or nucleic acid        fragments for their ability to induce production of antibodies        by the animal species against the unmodified APP or Aβ, and    -   identifying and optionally isolating the member(s) of the set of        analogues which significantly induces antibody production        against unmodified APP or Aβ in the species or identifying and        optionally isolating the polypeptide expression products encoded        by members of the set of nucleic acid fragments which        significantly induces antibody production against unmodified APP        or Aβ in the animal species.

In this context, the “set of mutually distinct modified amyloidogenicpolypeptides” is a collection of non-identical analouges which have e.g.been selected on the basis of the criteria discussed above (e.g. incombination with studies of circular dichroism, NMR spectra, and/orX-ray diffraction patterns). The set may consist of only a few membersbut it is contemplated that the set may contain several hundred members.The test of members of the set can ultimately be performed in vivo, buta number of in vitro tests can be applied which narrow down the numberof modified molecules which will serve the purpose of the invention.

Since the goal of introducing the foreign T-cell epitopes is to supportthe B-cell response by T-cell help, a prerequisite is that T-cellproliferation is induced by the analogue. T-cell proliferation can betested by standardized proliferation assays in vitro. In short, a sampleenriched for T-cells is obtained from a subject and subsequently kept inculture. The cultured T-cells are contacted with APCs of the subjectwhich have previously taken up the modified molecule and processed it topresent its T-cell epitopes. The proliferation of T-cells is monitoredand compared to a suitable control (e.g. T-cells in culture contactedwith APCs which have processed intact, native amyloidogenicpolypeptide). Alternatively, proliferation can be measured bydetermining the concentration of relevant cytokines released by theT-cells in response to their recognition of foreign T-cells.

Having rendered highly probable that at least one analogue of eithertype of set is capable of inducing antibody production against APP orAβ, it is possible to prepare an immunogenic composition comprising atleast one analouge which is capable of inducing antibodies againstunmodified APP or Aβ in an animal species where the unmodified APP or Aβis a self-protein, the method comprising admixing the member(s) of theset which significantly induces production of antibodies in the animalspecies which are reactive with the APP or Aβ with a pharmaceuticallyand immunologically acceptable carrier and/or vehicle and/or diluentand/or excipient, optionally in combination with at least onepharmaceutically and immunologically acceptable adjuvant.

The above-described tests of polypeptide sets are conveniently carriedout by initially preparing a number of mutually distinct nucleic acidsequences or vectors of the invention, inserting these into appropriateexpression vectors, transforming suitable host cells (or host animals)with the vectors, and effecting expression of the nucleic acid sequencesof the invention. These steps can be followed by isolation of theexpression products. It is preferred that the nucleic acid sequencesand/or vectors are prepared by methods comprising exercise of amolecular amplification technique such as PCR or by means of nucleicacid synthesis.

Specific Amyloidogenic Targets

In addition to the proteins most often associated with Alzheimer's, APP,ApoE4 and Tau, there is long list of other proteins that have somehowbeen linked to AD, either by their direct presence in plaques or tanglesof AD brains or by their apparent genetic association with increasedrisk of developing AD. Most, if not all, of these antigens are togetherwith the above-discussed Aβ, APP, presenilin and ApoE4, putative targetproteins in certain embodiment of the present invention. These putativetargets are already discussed thoroughly in WO 01/62284. Hence, theseputative targets will only be mentioned briefly here, whereas a morethorough background discussion can be be found in WO 01/62282 which ishereby incorporated by reference herein:

Alpha1-antichymotrypsin (ACT); Alpha2-macroglobulin; ABAD (Aβ-peptidebinding alcohol dehydrogenase); APLP1 and -2 (amyloid precursor likeprotein 1 and -2); AMY117; Bax; Bcl-2; Bleomycin hydrolase; BRI/ABRI;Chromogranin A; Clusterin/apoJ; CRF (corticotropin releasing factor)binding protein; EDTF (endothelial-derived toxic factor); Heparansulfate proteoglycans; Human collapsin response mediator protein-2;Huntingtin (Huntington's disease protein); ICAM-I; IL-6;Lysosome-associated antigen CD68; P21 ras; PLC-delta 1 (phospholipase Cisoenzyme delta 1); Serum amyloid P component (SAβ); Synaptophysin;Synuclein (alpha-synuclein or NACP); and TGF-b1 (transforming growthfactor b1).

The presently described means and methods for down-regulation of APP orAβ can be combined with therapies, e.g. active specific immunotherapy,against any of these other amyloidogenic polypeptides.

Apart from Alzheimer's disease, also cerebral amyloid angiopathy is adisease that would be a suitable target for the presently disclosedtechnology.

It is contemplated that most methods for immunizing against APP or Aβshould be restricted to immunization giving rise to antibodiescross-reactive with the native APP or Aβ. Nevertheless, in some cases itwill be of interest to induce cellular immunity in the form of CTLresponses against cells which present MHC Class I epitopes from theamyloidogenic polypeptides—this can be expedient in those cases whereinreduction in the number of cells producing APP or Aβ does not constitutea serious adverse effect. In such cases where CTL responses are desiredit is preferred to utilise the teachings of Applicant's WO 00/20027. Thedisclosures of these two documents are hereby incorporated by referenceherein.

Immunogen Carriers

Molecules comprising a T helper epitope and APP or Aβ peptidesrepresenting or including B-cell epitopes linked covalently to anon-immunogenic polymer molecule acting as a vehicle, e.g. a multivalentactivated poly-hydroxypolymer, will, as mentioned above, function as avaccine molecule that only contains the immunologically relevant parts,can be obtained, and are interesting embodiments in variants d and edisclosed above. Promiscuous or so-called universal T-helper epitopescan be used if e.g. the target for the vaccine is a self-antigen such asAPP or Aβ. Furthermore, elements that enhance the immunological responsecould be also co-coupled to the vehicle and thereby act as an adjuvant.Such elements could be mannose, tuftsin, muramyl dipeptide, CpG motifsetc. In that case, subsequent adjuvant formulation of the vaccineproduct might be unnecessary and the product could be administered inpure water or saline.

By coupling cytotoxic T cell (CTL) epitopes together with the T-helperepitopes it will also be possible to generate CTL's specific for theantigen from which the CTL epitope was derived. Elements that promoteuptake of the product to the cytosol, such as mannose, of the APC, e.g.a macrophage, could also be co-coupled to the vehicle together with theCTL- and the T helper epitope and enhance the CTL response.

The ratio of B-cell epitopes and T-helper epitopes (P2 and P30) in thefinal product can be varied by varying the concentration of thesepeptides in the synthesis step. As mentioned above, the immunogenicmolecule can be tagged with e.g. mannose, tuftsin, CpG-motifs or otherimmune stimulating substances (described herein) by adding these, ifnecessary by using e.g. aminated derivatives of the substances, to thecarbonate buffer in the synthesis step.

If an insoluble activated polyhydroxy polymer is used to combine thepeptides containing the APP or Aβ B-cell epitope and the T-helperepitopes it can, as mentioned above be performed as a solid phasesynthesis and the final product can be harvested and purified by washand filtration. The elements to be coupled to a tresyl activatedpolyhydroxypolymer (peptides, tags etc) can be added to thepolyhydroxypolymer at low pH, e.g. pH 4-5, and allowed to be equallydistributed in the “gel” by passive diffusion. Subsequently, the pH canbe raised to pH 9-10 to start the reaction of the primary amino groupson the peptides and tags to the tresyl groups on the polyhydroxypolymer. After coupling of peptides and e.g. immune stimulating elementsthe gel is grinded to form particles of suitable size for immunization.

Such an immunogen therefore comprises

-   -   a) at least one first amino acid sequence derived from APP or        Aβ, wherein the at least one first amino acid sequence contains        at least one B-cell and/or at least one CTL epitope, and    -   b) at least one second amino acid sequence that includes a        foreign T helper cell epitope,        wherein each of the at least first and at least second amino        acid sequences are coupled to a pharmaceutically acceptable        activated polyhydroxypolymer carrier.

In order for the amino acid sequences to couple to thepolyhydroxypolymer it is normally necessary to “activate” thepolyhydroxypolymer with a suitable reactive group that can form thenecessary link to the amino acid sequences.

The term “polyhydroxypolymer” is intended to have the same meaning as inWO 00/05316, i.e. the polyhydroxypolymer can have exactly the samecharacteristics as is specifically taught in that application. Hence,the polyhydroxypolymer can be water soluble or insoluble (thus requiringdifferent synthesis steps during preparation of the immunogen). Thepolyhydroxypolymer can be selected from naturally occurring polyhydroxycompounds and synthetic polyhydroxy compounds.

Specific and preferred polyhydroxypolymers are polysaccharides selectedfrom acetan, amylopectin, gum agar-agar, agarose, alginates, gum Arabic,carregeenan, cellulose, cyclodextrins, dextran, furcellaran,galactomannan, gelatin, ghatti, glucan, glycogen, guar, karaya,konjac/A, locust bean gum, mannan, pectin, psyllium, pullulan, starch,tamarine, tragacanth, xanthan, xylan, and xyloglucan. Dextran isespecially preferred.

However, the polyhydroxypolymer can also be selected from highlybranched poly(ethyleneimine)(PEI), tetrathienylene vinylene, Kevlar(long chains of poly-paraphenyl terephtalamide), Poly(urethanes),Poly(siloxanes), polydimethylsiloxane, silicone, Poly(methylmethacrylate) (PMMA), Poly(vinyl alcohol), Poly(vinyl pyrrolidone),Poly(2-hydroxy ethyl methacrylate), Poly(N-vinyl pyrrolidone),Poly(vinyl alcohol), Poly(acrylic acid), Polytetrafluoroethylene (PTFE),Polyacrylamide, Poly(ethylene-co-vinyl acetate), Poly(ethylene glycol)and derivatives, Poly(methacrylic acid), Polylactides (PLA),Polyglycolides (PGA), Poly(lactide-co-glycolides) (PLGA),Polyanhydrides, and Polyorthoesters.

The (weight) average molecular weight of the polyhydroxypolymer inquestion (i.e. before activation) is typically at least 1,000, such asat least 2,000, preferably in the range of 2,500-2,000,000, morepreferably in the range of 3,000-1,000,000, in particular in the rangeof 5,000-500,000. It has been shown in the examples thatpolyhydroxypolymers having an average molecular weight in the range of10,000-200,000 are particularly advantageous.

The polyhydroxypolymer is preferably water soluble to an extent of atleast 10 mg/ml, preferably at least 25 mg/ml, such as at least 50 mg/ml,in particular at least 100 mg/ml, such as at least 150 mg/ml at roomtemperature. It is known that dextran, even when activated as describedherein, fulfils the requirements with respect to water solubility.

For some of the most interesting polyhydroxypolymers, the ratio betweenC (carbon atoms) and OH groups (hydroxy groups) of the unactivatedpolyhydroxypolymers (i.e. the native polyhydroxypolymer beforeactivation) is in the range of 1.3 to 2.5, such as 1.5-2.3, preferably1.6-2.1, in particular 1.85-2.05. Without being bound to any specifictheory, it is believed that such as a C/OH ratio of the unactivatedpolyhydroxypolymer represents a highly advantageous level ofhydrophilicity. Polyvinylalcohol and polysaccharides are examples ofpolyhydroxypolymers which fulfil this requirement. It is believed thatthe above-mentioned ratio should be roughly the same for the activatedpolyhydroxypolymer as the activation ratio should be rather low.

The term “polyhydroxypolymer carrier” is intended to denote the part ofthe immunogen that carries the amino acid sequences. As a general rule,the polyhydroxypolymer carrier has its outer limits where the amino acidsequences can be cleaved of by a peptidase, e.g. in an antigenpresenting cell that is processing the immunogen. Hence, thepolyhydroxypolymer carrier can be the polyhydroxypolymer with anactivation group, where the bond between the activation group and theamino acid sequence is cleavable by a peptidase in an APC, or thepolyhydroxypolymer carrier can be a polyhydroxypolymer with activationgroup and e.g. a linker such as a single L-amino acid or a number ofD-amino acids, where the last part of the linker can bond to the aminoacid sequences and be cleaved by a peptidase in an APC.

As mentioned above, the polyhydroxypolymers carry functional groups(activation groups), which facilitate the anchoring of peptides to thecarrier. A wide range of applicable functional groups are known in theart, e.g. tresyl (trifluoroethylsulphonyl), maleimido, p-nitrophenylcloroformate, cyanogenbromide, tosyl (p-toluenesulfonyl), triflyl(trifluoromethanesulfonyl), pentafluorobenzenesulfonyl, and vinylsulphone groups. Preferred examples of functional groups within thepresent invention are tresyl, maleimido, tosyl, triflyl,pentafluorobenzenesulfonyl, p-nitrophenyl cloroformate, andvinylsulphone groups, among which tresyl, maleimido, and tosyl groupsare particularly relevant.

Tresyl activated polyhydroxypolymers can be prepared using tresylchloride as described for activation of dextran in Example 1 in WO00/05316 or as described in Gregorius et al., J. Immunol. Meth. 181(1995) 65-73.

Maleimido activated polyhydroxypolymers can be prepared usingp-maleimidophenyl isocyanate as described for activation of dextran inExample 3 of WO 00/05316. Alternatively, maleimido groups could beintroduced to a polyhydroxypolymer, such as dextran, by derivatisationof a tresyl activated polyhydroxypolymer (such as tresyl activateddextran (TAD)) with a diamine compound (generally H₂N—C_(n)H_(2n)—NH₂,where n is 1-20, preferably 1-8), e.g. 1,3-diaminopropane, in excess andsubsequently react the amino groups introduced in TAD with reagents suchas succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),sulfo-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-SMCC), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB),sulfo-succinimidyl 4-(p-maleimidophenyl)butyrate (sulfo-SMPB),N-γ-maleimidobutyryloxy-succinimide ester (GMBS) orN-γ-maleimidobutyryloxy-sulfosuccinimide ester. Although the differentreagents and routes for activation formally results in slightlydifferent maleimide activated products with respect to the linkagebetween the maleimide functionality and the remainder of the parenthydroxy group on which activation is performed, all and every areconsidered as “maleimide activated polyhydroxypolymers”.

Tosyl activated polyhydroxypolymers can be prepared using tosyl chlorideas described for activation of dextran in Example 2 in WO 00/05316.Triflyl and pentafluorobenzenesulfonyl activated polyhydroxypolymers areprepared as the tosyl or tresyl activated analogues, e.g. by using thecorresponding acid chlorides.

Cyanogenbromide activated polyhydroxypolymer can be prepared by reactingthe polyhydroxypolymer with cyanogenbromide using conventional methods.The resulting functional groups are normally cyanate esters with twohydroxy groups of the polyhydroxypolymer.

The degree of activation can be expressed as the ratio between the freehydroxy groups and the activation groups (i.e. functionalised hydroxygroups). It is believed that a ratio between the free hydroxy groups ofthe polyhydroxypolymer and the activation groups should be between 250:1and 4:1 in order to obtain an advantageous balance between thehydrophilicity and the reactivity of the polyhydroxypolymer. Preferablythe ratio is between 100:1 and 6:1, more preferably between 60:1 and8:1, in particular between 40:1 and 10:1.

Especially interesting activated polyhydroxypolymers for use in themethod for producing the generally applicable immunogen according to theinvention are tresyl, tosyl and maleimido activated polysaccharides,especially tresyl activated dextran (TAD), tosyl activated dextran(TosAD), and maleimido activated dextran (MAD).

It is preferred that the bond between the polyhydroxypolymer carrier andthe amino acid sequences attached thereto are cleavable by a peptidase,e.g. as a peptidase active in the processing of antigens in an APC. Itis therefore preferred that the at least first and at least second aminoacid sequences are coupled to the activated polyhydroxypolymer carriervia an amide bond or a peptide bond. It is especially preferred that theat least first and at least second amino acid sequences each provide forthe nitrogen moiety of their respective amide bond.

The polyhydroxypolymer carrier may be substantially free of amino acidresidues, necessitating that the activation group provides for part of apeptidase cleavable bond, but as mentioned above, the carrier may alsosimply include a spacer including at least one L-amino acid.Nevertheless, the at least first and at least second amino acidsequences are normally bound to the activated version of thepolyhydroxypolymer via the nitrogen at the N-terminus of the amino acidsequence.

The above-described generally applicable immunogen of the presentinvention can be used in immunization methods essentially as describedherein for polypeptide vaccines. That is, all disclosures relating todosages, mode of administration and formulation of polypeptide vaccinesfor down-regulating the amyloidogenic polypeptides discussed hereinapply mutatis mutandis to the generally applicable immunogens.

Generally Applicable Safe Vaccination Technology

As discussed above, one preferred embodiment of the present inventionentails the use of variants of amyloidogenic polypeptides that areincapable of providing self-derived T_(H) epitopes that may drive animmune response against the amyloidogenic polypeptide.

However, it is believed by the present inventors that this strategy fordesigning anti-self vaccines and for effecting anti-self immunity, is agenerally applicable technology that is inventive in its own right. Itshould prove especially suited in cases where the self-antigen it issought to down-regulate is sufficiently abundant in the body so that itis possible that self-stimulation of an immune response could happen.Hence, all disclosures above of this embodiment insofar as it relates tothe provision of an anti-self immune response against APP or Aβ appliesmutatis mutandis to immunization against other self-polypeptides,especially those that are present in sufficient amounts for them tomaintain the immune response in the form of an uncontrolled autoimmunecondition because autologous T_(H) epitopes of the relevantself-polypeptide are driving the immune response.

EXAMPLE 1

The Auto Vaccination Approach for Immunizing Against AD

The fact that Aβ protein knock out mice does not show any abnormalitiesor adverse side effects, suggest that removal or lowering the amounts ofAβ will be safe, Zheng H. (1996).

Published experiments where transgenic animals are immunized against thetransgenic human Aβ protein suggest that if it was possible to break theself tolerance, down-regulation of Aβ could be obtained by auto-reactiveantibodies. These experiments further suggest that such down regulationof Aβ potentially would both prevent the formation of plaques, and evenclear already formed Aβ plaques from the brain, cf. Schenk et al.(1999). But, traditionally it is not possible to raise antibodiesagainst self-proteins.

The published data does thus not provide the means for breaking trueself-tolerance towards true self-proteins. Nor does the data provideinformation on how to ensure that the immune reaction is directed solelyor predominantly towards the Aβ deposits, and not towards the cellmembrane bound Aβ precursor protein (APP), if this is deemed necessary.An immune response generated using the existing technology wouldpresumably generate an immune response towards self-proteins in anunregulated way so unwanted and excessive auto-reactivity towards partsthe Aβ protein may be generated. Hence, using existing immunizationstrategies will most likely be unable to generate strong immuneresponses towards self-proteins and will furthermore be unsafe due topotential strong cross-reactivity towards membrane bound APP which ispresent on a large number of cells in the CNS.

The present invention provides the means of effectively generating astrong regulated immune response towards true self-proteins whichpotentially could form plaques and cause serious disease in the CNS orin other compartments of the body. A safe and efficacious human Aβprotein therapeutic vaccine will be developed by using this technologyfor the treatment of AD.

In light of this, it is possible to anticipate that AD, a diseasepredicted to cripple the health care system in the next century, couldbe cured, or such vaccines described could at least constitute aneffective therapeutical approach for treatment of the symptoms andprogression of this disease. This technique represents a entirely newimmunological approach to blocking amyloid deposition in AD and otherneurologic diseases as well.

In the following table, 35 contemplated constructs are indicated. Allpositions given in the table are relative to the starting Methionine ofAPP (first amino acid in SEQ ID NO: 2) and include both the starting andending amino acid, e.g. the 672-714 fragment includes both amino acid672 and 714. The starting and ending positions for P2 and P30 indicatethat the epitope substitutes a part of the APP fragment at the positionsindicated (both positions included in the substitution)—in mostconstructs, the introduced epitopes substitutes a fragment of the lengthof the epitope. The asterisks in the table have the following meaning:APP AutoVac constructions Start of End of Position of Position of APPsegment APP segment P2 epitope P30 epitope relative to relative torelative to relative to Molecule Var. No. aa 1 of APP aa 1 of APP aa 1of APP aa 1 of APP length 1 630 770 656-670 635-655 141 2 630 714656-670 635-655 85 3 672 770 735-749 714-728 99 4 672 770 714-728 99 5672 770 714-728 99 6 672 770 723* 723* 135 7 672 770 723* 120 8 672 770723* 114 9 672 714 672* 64 10 672 714 714* 64 11 672 714 672* 58 12 672714 714* 58 13 672 714 714* 672* 79 14 672 714 680-694 43 14 672 714685-799 43 16 672 714 690-704 43 17 672 714 695-709 43 18 672 714675-695 43 19 672 714 680-700 43 20 672 714 685-705 43 21 672 714690-710 43 22 672 714 680* 680* 79 23 672 714 690* 690* 79 24 672 714700* 700* 79 25 672 714 710* 710* 79 26 672 714 680* 64 27 672 714 690*64 28 672 714 700* 64 29 672 714 710* 64 30 672 714 680* 58 31 672 714690* 58 32 672 714 700* 58 33 672 714 710* 58 34 672 714 After rep. 1**After rep. 2** 165 35 672 714 34 × 3* 34 × 3*** 165*Only one position for P2 and P30 indicates that the epitope has beeninserted into the APP derivative at the position indicated (the epitopebegins at the amino acid C-terminally adjacent to the given position).**Construction 34 contains three identical APP fragments separated byP30 and P2, respectively.***Construction 35 contains nine identical APP fragments separated byalternating P30 and P2 epitopes.

The part of APP, against which it most interesting to generate aresponse, is the 43 amino acid Aβ core peptide (Aβ-43, corresponding toSEQ ID NO: 2, residues 672-714) that is the main constituent of amyloidplaques in AD brains. This APP fragment is part of all constructionslisted above.

Variants 1 and 2 comprise a portion of APP upstream of Aβ-43 where themodel epitopes P2 and P30 have been placed. Variants 1 and 3-8 allcomprise the C-100 fragment which has been shown to be neurotoxic—theC-100 fragment corresponds to amino acid residues 714-770 of SEQ ID NO:2. In variants 3-5 the epitopes substitutes a part of the C-100 fragmentwhile the in variants 6-8 have been inserted into C-100.

Variants 9-35 contain only the core Aβ-43 protein. In variants 9-13, P2and P30 are fused to either end of Aβ-43; in 14-21 P2 and P30substitutes part of Aβ-43; in 22-33 P2 and P30 are inserted into Aβ-43;34 contains three identical Aβ-43 fragments spaced by P30 and P2,respectively; 35 contains 9 Aβ-43 repeats spaced by alternating P2 andP30 epitopes.

Truncated parts of the above-discussed Aβ-43 protein can also beemployed in immunogenic analogues according to the present invention.Especially preferred are the truncates Aβ(1-42), Aβ(1-40), Aβ(1-39),Aβ(1-35), Aβ(1-34), Aβ(1-34), Aβ(1-28), Aβ(1-12), Aβ(1-5), Aβ(13-28),Aβ(13-35), Aβ(17-28), Aβ(25-35), Aβ(35-40), Aβ(36-42), and Aβ(35-42)(where the numbers in the parentheses indicate the amino acid stretchesof Aβ-43 that constitute the relevant fragment—Aβ(35-40) is e.g.identical to amino acids 706-711 in SEQ ID NO: 2). All these variantswith truncated parts of Aβ-43 can be made with the Aβ fragmentsdescribed herein, in particular with variants 9, 10, 11, 12, and 13.

In some cases, it is preferred that the Aβ-43 or fragments thereof aremutated. Especially preferred are substitution variants where themethionine in position 35 in Aβ-43 has been substituted, preferably withleucine or isoleucine, or simply deleted. Especially preferred analoguescontain one single methionine that is located in the C-terminus, eitherbecause it is naturally occurring in the amyloidogenic polypeptide orforeign T_(H) epitope, or because it has been inserted or added. Hence,it also preferred that the part of the analogue that includes theforeign T_(H) epitope is free from methionine, except from the possibleC-terminal location of a methionine.

In fact, it is generally preferred that all analogues of APP or Aβ thatare used according to the present invention share the characteristic ofmerely including one single methionine that is positioned as theC-terminal amino acid in the analogue and that other methionines ineither the amyloidogenic polypeptide or the foreign T_(H) epitope aredeleted or substituted for another amino acid.

One further interesting mutation is a deletion or substitution of thephenylalanine in position 19 in Aβ-43, and it is especially preferredthat the mutation is a substitution of this phenylalanine residue with aproline.

The following table sets forth a group of especially preferredconstructs that operate with truncates or mutations of Aβ-43: Positionof Position of P2 epitope P30 epitope Total Aβ segment used in Positionof Aβ segment relative to relative to length Variant molecule relativeto relative to aa 1 of aa 1 of aa 1 of of molecule No. aa 1 of Aβ(1-42/43) molecule molecule molecule (aa) 36 1-28 22-49 50-64  1-21 6437 1-12 (a) + 13-28 (b) 1-12 (a) + 49-64 (b) 34-48 13-33 64 38 1-12 (×3) 1-12, 34-45, 61-72 46-60 13-33 72 39 13-28 (× 3) 1-16, 38-53, 69-8454-68 17-37 84 40 1-12 (a) + 13-35 (b) + 36-42 1-12 (a) + 34-56 (b) +72-78 57-71 13-33 78 (c) (c) 41 1-28 (× 3) 1-28, 50-77, 93-120 78-9229-49 120 42 1-43 (F19P/M35K) 1-43 65-79 44-64 79

In this table, the Aβ segment used in the molecule is indicated by aminoacid numbers relative to aa 1 of the Aβ(1-42/43) molecule, i.e. 1-28means that fragment 1-28 of Aβ(1-42/43) is used in the molecule. If twoor more different segments are used, both are indicated in the table,i.e. 1-12 (a)+13-28 (b) means that both fragment 1-12 and fragment 13-28of Aβ(1-42/43) are used in the molecule.

Also, if the same segment is present in more than one copy in theconstruction it is indicated in the table, i.e. 1-12 (×3) shows thatfragment 1-12 of Aβ(1-42/43) is present in three copies in theconstruction.

Further, the position of the Aβ segment in the molecule is shown byamino acid positions relative to the first amino acid of the molecule,i.e. 22-49 shows that the Aβ fragment in question is positioned fromamino acid 22 to amino acid 49 in the molecule, both positions included.Positions of the P2 and P30 epitopes are indicated equivalently. If twoor more different Aβ fragments are used in the molecule, their positionsare all shown, i.e. 1-12 (a)+49-64 (b) means that fragment (a) ispositioned from aa 1-12 in the molecule and fragment (b) from aa 49-64.

Moreover, if more than one copy of the same fragment is present in themolecule, positions for all copies are shown, i.e. 1-12, 34-45, 61-72shows that the three copies of the Aβ fragment are placed from position1-12, 34-45 and 61-72, respectively, in the molecule.

Finally, the total length indication of each molecule includes both theAβ fragment(s) and the P2 and P30 epitopes.

Variant 42 contains two amino acid substitutions at positions 19 (phe topro) and 35 (met to lys) as it is indicated in the column showing the Aβfragments.

See FIG. 1 and the tables above for details on particular points forintroduction of the foreign T-cell epitopes.

One further type of construct is especially preferred. Since one goal ofthe present invention is to avoid destruction of the cells producing APPwhereas removal of Aβ is desired, it seems feasible to prepareautovaccine constructs comprising only parts of Aβ which are not exposedto the extracellular phase when present in APP. Thus, such constructswould need to contain at least one B-cell epitope derived from the aminoacid fragment defined by amino acids 700-714 in SEQ ID NO: 2. Since sucha short polypeptide fragment is predicted to be only weakly immunogenicit is preferred that such an autovaccine construct consists of severalcopies of the B-cell epitope, e.g. in the form of a construct having thestructure shown in Formula I in the detailed disclosure of the presentinvention, cf. above. In that version of Formula I, the termsamyloid_(e1)-amyloid_(ex) are x B-cell epitope containing amino acidsequences derived from amino acids 700-714 of SEQ ID NO: 2. A preferredalternative is the above-detailed possibility of coupling theamyloidogenic (poly)peptide and the selected foreign T-helper epitope tovia an amide bond to a polysaccharide carrier molecule—in this waymultiple presentaions of the “weak” epitope constituted by amino acids700-714 of SEQ ID NO: 2 become possible, and it also becomes possible toselect an optimum ratio between B-cell and T-cell epitopes.

EXAMPLE 2

Immunisation of Transgenic Mice with Aβ and Modified Proteins Accordingto the Invention

Construction of the hAB43+-34 encoding DNA. The hAB43+-34 gene wasconstructed in several steps. First a PCR fragment was generated withprimers ME#801 (SEQ ID NO: 10) and ME#802 (SEQ ID NO: 11) using primerME#800 (SEQ ID NO: 9) as template. ME#800 encodes the human abeta-43fragment with E. coli optimised codons. ME#801 and 802 adds appropriaterestriction sites to the fragment.

The PCR fragment was purified, digested with NcoI and HindIII, purifiedagain and cloned into NcoI-HindIII digested and purified pET28b+E. coliexpression vector. The resulting plasmid encoding wildtype human Aβ-43is named pAB1.

In the next step the T-helper epitope, P2, is added to the C-terminus ofthe molecule. Primer ME#806 (SEQ ID NO: 12) contains the sequenceencoding the P2 epitope, thus generating a fusion of P2 and Abeta-43 bythe PCR reaction.

The cloning was performed by making a PCR fragment with primers ME#178(SEQ ID NO: 8) and ME#806 using pAB1 as template. The fragment waspurified, digested with NcoI and HindIII, purified again and cloned intoan NcoI-HindIII digested and purified pET28b+ vector. The resultingplasmid is called pAB2.

In an analogous manner, another plasmid was made harbouring the Aβ-43encoding sequence with another T helper epitope, P30, added to theN-terminus. This was done by making a PCR fragment with primers ME#105(SEQ ID NO: 7) and ME#807 (SEQ ID NO: 13) using pABl as template.

The fragment was purified, digested with NcoI and HindIII, purifiedagain and cloned into an NcoI-HindIII digested and purified pET28b+vector. The resulting plasmid is called pAB3.

In the third step, a second Aβ-43 repeat is added C-terminally to the P2epitope of plasmid pAB2 by primer ME#809 (SEQ ID NO: 14). ME#809 at thesame time creates a BamHI site immediately after the Aβ-43 repeat. A PCRfragment was made with primers ME#178 and ME#809 using pAB2 as template.The fragment was digested with NcoI and HindIII, purified and clonedinto NcoI-HindIII digested and purified pET28b+ vector. This plasmid isnamed pAB4.

Finally, the P30 epitope—Aβ-43 repeat sequence from pAB3 was cloned intopAB4 plasmid. This was done by making a PCR fragment with primers ME#811(SEQ ID NO: 16) and ME#105 using pAB3 as template. The fragment waspurified and used as primer in a subsequent PCR with ME#810 (SEQ ID NO:15) using pAB3 as template. The resulting fragment was purified,digested with BamHI and HindIII and cloned into BamHI-HindIII digestedand purified pAB4 plasmid. The resulting plasmid, pAB5, encodes thehAB43+-34 molecule.

All PCR and cloning procedures were done essentially as described bySambrook, J., Fritsch, E. F. & Maniatis, T. 1989 “Molecular cloning: alaboratory manual”. 2nd. Ed. Cold Spring Harbor Laboratory, N.Y.

For all cloning procedures E. coli K-12 cells, strain Top-10 F′(Stratagene, USA), were used. The pET28b+ vector was purchased fromNovagen, USA. All primers were synthesised at DNA Technology, Denmark.

Expression and purification of hAB43+-34. The hAB43+-34 protein encodedby pAB5 was expressed in BL21-Gold (Novagen) E. coli cells as describedby the suppliers of the pET28b+ system (Novagen).

The expressed hAB43+-34 protein was purified to more than 85% purity bywashing of inclusion bodies followed by cation-exchange chromatographyusing a BioCad purification workstation (PerSeptive Biosystems, USA) inthe presence of 6 M urea. The urea was hereafter removed by stepwisedialysis against a solution containing decreasing amounts of urea. Thefinal buffer was 10 mM Tris, pH 8.5.

Immunisation study. Mice transgenic for human APP (Alzheimer's precursorprotein) were used for the study. These mice, called TgRND8+, express amutated form of APP that results in high concentration of Aβ-40 andAβ-42 in the mouse brains (Janus, C. et. al.)

The mice (8-10 mice per group) were immunised with either Abeta-42 (SEQID NO: 2, residues 673-714, synthesised by means of a standard Fmocstrategy) or the hAB43+-34 variant (construct 34 in the table in Example1, recombinantly produced) four times at two-week intervals. Doses wereeither 100 mg for Aβ or 50 mg for hAB43+-34. Mice were bled at day 43(after three injections) and after day 52 (after four injections) andthe sera were used to determine the level of anti-Aβ-42 specific titresusing a direct Aβ-42 ELISA.

The following tabel shows the mean relative anti-Abeta-42 titres. Day 43Immunogen (after 3 immunizations) Day 52 (after 4 immunizations) Aβ-424000 3000 hAB43+−34 16000 23000

As will be clear, the antibody titers obtained when immunizing with thehAB43+-34 Aβ variant are approximately 4 times and 7.5 times higherafter 3 and 4 immunizations, respectively, than the titers obtained whenusing the unaltered wild-type Aβ-42 as an immunogen. This fact is putfurther in perspective, when considering the fact that the amount ofvariant used for immunization was only 50% of the amount of wild-typesequence used for immunization.

EXAMPLE 3

Synthesis of an Aβ Peptide Copolymer Vaccine Using ActivatedPoly-hydroxypolymer as the Cross-linking Agent.

Introduction. A traditional conjugate vaccine consists of a(poly)peptide coupled covalently to a carrier protein. The peptidecontains the B-cell epitope(s) and the carrier protein provides T-helperepitopes. However, most of the carrier protein will normally beirrelevant as a source for T-helper epitopes, since only a minor part ofthe total sequence contains the relevant T-helper epitopes. Suchepitopes can be defined and synthesized as peptides of e.g. 12-15 aminoacids. If these peptides are linked covalently to peptides containingthe B-cell epitopes, e.g. via a multivalent activatedpoly-hydroxypolymer, a vaccine molecule that only contains the relevantparts can be obtained. It is further possible to provide a vaccineconjugate that contains an optimized ratio between B-cell and T-cellepitopes.

Synthesis of the acticated poly-hydrocypolymer. Poly-hydroxypolymerssuch as dextran, starch, agarose etc. can be activated with2,2,2-trifluoroethanesulfonyl chloride (tresyl chloride), either bymeans of a homogenous synthesis (dextran) dissolved inN-methylpyrrolidinone (NMP) or by means of a heterogeneous synthesis(starch, agarose, cross-linked dextran) in e.g. acetone.

225 ml dry N-methyl pyrrolidinone (NMP) is added under dry conditions tofreeze dried, water-soluble dextran (4.5 g, 83 mmol, clinical grade,Mw(avg) 78000) in a 500 ml round bottom flask supplied with a magnet forstirring. The flask is placed in a 60° C. oil bath with magneticstirring. The temperature is raised to 92° C. over a period of 20 min.When the dextran is dissolved the flask is immediately removed from theoil bath and the temperature in the bath is lowered to 40° C. The flaskis placed into the oil bath agaom, still with magnetic stirring, andtresyl chloride (2.764 ml, 25 mmol) is added drop-wise. After 15 min,dry pyridine (anhydrous, 2.020 ml, 25 mmol) is added drop-wise. Theflask is removed from the oil bath and stirred for 1 hour at roomtemperature. The product (Tresyl Activated Dextran, TAD) is precipitatedin 1200 ml cold ethanol (99.9%). The supernatant is decanted and theprecipitate is harvested in 50 ml polypropylene tubes in a centrifuge at2000 rpm. The precipitate is dissolved in 50 ml 0.5% acetic acid,dialyzed 2 times against 5000 ml 0.5% acetic acid and freeze dried. TADcan be stored as a freeze dried powder at −20° C.

An insoluble poly-hydroxypolymer, such as agarose or croos-linkeddextran can be tresyl activated by making a suspension of thepoly-hydroxypolymer in e.g. acetone and perform the synthesis as a solidphase synthesis. The activated poly-hydroxypolymer can be harvested byfiltration. Suitable methods are reported in e.g. Nilsson K and MosbachK (1987), Methods in Enzymology 135, p. 67, and in Hermansson G T et al.(1992), in “Immobilized Affinity Ligand Techniques”, Academic Press,Inc., p. 87.

Synthesis of the A Beta Peptide Copolymers Vaccines. TAD (10 mg) isdissolved in 100 μl H₂O and 1000 μl carbonate buffer, pH 9.6, containing5 mg Aβ-42 (SEQ ID NO: 2, residues 673-714), 2.5 mg P2 (SEQ ID NO: 4)and 2.5 mg P30 (SEQ ID NO: 6) is added. The Aβ-42 and the P2 and P30peptides all contain protected lysine groups: these are in the form of1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) protected lysinegroups. The peptides are prepared by means of a standard Fmoc strategy,where the conventional Fmoc-Lys(Boc)-OH has been substituted withFmoc-Lys(Dde)-OH (obtained from Novabiochem , cat. no. 04-12-1121), i.e.the e-amino group in lysine is protected with Dde instead of Boc.

The pH value is measured and adjusted to 9.6 using 1 M HCl. After 2.5hours at room temperature, hydrazine from an 80% solution is added to afinal hydrazine koncentration of 8% and the solution is incubated foranother 30 min. at room temperature and freeze-dried immediatelyhereafter. The freeze-dried product is dissolved in H₂O and dialysedextensively against H₂O before the final freeze-drying. The ratiobetween B-cell epitopes (Aβ) and T-helper epitopes (P2 and P30) in thefinal product can be varied by using different concentrations of thesepeptides in the synthesis step. Furhermore, the final product can betagged with e.g. mannose (so as to target the conjugate to APCs) byadding aminated mannose to the carbonate buffer in the synthesis step.

If an insoluble activated poly-hydroxypolymer is used to combine thepeptides containing the B-cell epitope and the T-helper epitopes, thecoupling to the polymer can be performed as a solid phase synthesis andthe final product is harvested and purified by wash and filtration.

As mentioned in the general description, the presently describedapproach for preparing a peptide based vaccine may be applied to anyother polypeptide antigen where it would be convenient to prepare apurely synthetic peptide vaccine and where the polypeptide antigen inquestion provides a sufficient immunogenicity in one single peptide:

EXAMPLE 4

Synthesis Peptide Copolymer Vaccines

TAD (10 mg) is dissolved in 100 μl H₂O and 1000 μl carbonate buffer, pH9.6, containing 1-5 mg peptide A (any immunogenic peptide of interest!),1-5 mg P2 (diphtheria toxoid P2 epitope) and 1-5 mg P30 (diphtheriatoxoid P30 epitope) is added. The pH value is measured and adjusted to9.6 using 0.1 M HCl. After 2.5 hours at room temperature the solution isfreeze dried immediately hereafter. The freeze-dried product isdissolved in H₂O and dialysed extensively against H₂O or desalted on agelfiltration column before the final freeze-drying. In case thepeptides have lysine in the sequence the ε-amine in the lysine sidechain should be protected by Dde using the Fmoc-Lys(Dde)-OH derivativein the synthesis (Gregorius and Theisen 2001, submitted). Aftercoupling, hydrazine from an 80% solution is added to a final hydrazineconcentration between 1-20% and the solution is incubated for another 30min at room temperature, freeze dried immediately hereafter and dialysedextensively against H₂O or desalted on a gelfiltration column before thefinal freeze-drying. The principle is set forth in schematic form inFIG. 2.

Such immunogens have been utilised by the inventors with a shortC-terminal fragment of the Borrelia burgdorferi protein OspC as “peptideA” and a diptheria toxoid epitope (P2 or P30) as a peptide B. Theresults of immunization studies with this antigen revealed that only theimmunogen of the invention including the OspC fragment and a foreigndiptheria epitope matching the MHC haplotype of the vaccinated mice werecapable of inducing antibodies reactive with OspC in these mice. Incontrast, a molecule containing only the OspC peptide was unable toinduce antibody production and the same was true for a mixture of 2immunogens where one contained the OspC and the other the epitope. It istherefore concluded that the inclusion in the same polyhydroxypolymercarrier is superior, if not essential, in order to induce antibodyproduction against a short peptide hapten as OspC.

The invention will now be further described by the following numberedparagraphs:

-   -   1. A method for in vivo down-regulation of amyloid precursor        protein (APP) or beta amyloid (Aβ) in an animal, including a        human being, the method comprising effecting presentation to the        animal's immune system of an immunogenically effective amount of        at least one analogue of APP or Aβ that incorporates into the        same molecule a substantial fraction of B-cell epitopes of APP        and/or Aβ so that the analogue reacts to the same extent as does        APP or Aβ with a polyclonal serum raised against APP or Aβ, and        at least one foreign T-helper epitope (T_(H) epitope) so that        immunization of the animal with the analogue induces production        of antibodies against the animal's autologous APP or Aβ, wherein        the analogue        -   a) is a polyamino acid that contains the at least one            foreign T_(H) epitope and a disrupted APP or Aβ sequence so            that the analogue does not include any subsequence of SEQ ID            NO: 2 that binds productively to MHC class II molecules            initiating a T-cell response; and/or        -   b) is a conjugate comprising a polyhydroxypolymer backbone            to which is separately coupled a polyamino acid as defined            in a); and/or        -   c) is a conjugate comprising a polyhydroxypolymer backbone            to which is separately coupled 1) the at least one foreign            T_(H) epitope and 2) a disrupted sequence of APP or Aβ as            defined in a).    -   2. The method according to paragraph 1, wherein        -   at least one first moiety is introduced which effects            targeting of the analogue to an antigen presenting cell            (APC) or a B-lymphocyte, and/or        -   at least one second moiety is introduced which stimulates            the immune system, and/or        -   at least one third moiety is introduced which optimizes            presentation of the analogue to the immune system.    -   3. The method according to paragraph 2, wherein the first and/or        the second and/or the third moiety is/are attached as side        groups by covalent or non-covalent binding to suitable chemical        groups in the APP or Aβ sequence.    -   4. The method according to any one of the preceding paragraphs,        wherein the analogue comprises a fusion polypeptide.    -   5. The method according to any one of the preceding paragraphs,        wherein the analogue includes duplication of at least one B-cell        epitope of APP or Aβ and/or introduction of a hapten.    -   6. The method according to any one of the preceding paragraphs,        wherein the foreign T-cell epitope is immunodominant in the        animal.    -   7. The method according to any one of the preceding paragraphs,        wherein the foreign T-cell epitope is promiscuous, such as a        foreign T-cell epitope which is selected from a natural        promiscuous T-cell epitope and an artificial MHC-II binding        peptide sequence.    -   8. The method according to paragraph 7, wherein the natural        T-cell epitope is selected from a Tetanus toxoid epitope such as        P2 or P30, a diphtheria toxoid epitope, an influenza virus        hemagluttinin epitope, and a P. falciparum CS epitope.    -   9. The method according to any one of the preceding paragraphs,        wherein the analogue comprises B-cell epitopes which are not        exposed to the extracellular phase when present in a cell-bound        form of the precursor polypeptide Aβ.    -   10. The method according to any one of the preceding paragraphs,        wherein the analogue lacks at least one B-cell epitope which is        exposed to the extracellular phase when present in a cell-bound        form of the precursor polypeptide.    -   11. The method according to any one of the preceding paragraphs,        wherein the analogue comprises at most 9 consecutive amino acids        of SEQ ID NO: 2., such as at most 8, at most 7, at most 6, at        most 5, at most 4, and at most 3 consecutive amino acids.    -   12. The method according to paragraph 11, wherein the analogue        comprises at least one subsequence of SEQ ID NO: 2 so that each        such at least one subsequence of SEQ ID NO: 2 independently        consists of amino acid stretches selected from the group        consisting of 9 consecutive amino acids of SEQ ID NO: 2, 8        consecutive amino acids of SEQ ID NO: 2, 7 consecutive amino        acids of SEQ ID NO: 2, 6 consecutive amino acids of SEQ ID NO:        2, 5 consecutive amino acids of SEQ ID NO: 2, 4 consecutive        amino acids of SEQ ID NO: 2, and 3 consecutive amino acids of        SEQ ID NO: 2.    -   13. The method according to paragraph 11 or 12, wherein the        consecutive amino acids begin at an amino acid residue selected        from the group consisting of residue 672, 673, 674, 675, 676,        677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689,        690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702,        703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, and 714.    -   14. The method according to any one of the preceding paragraphs,        wherein presentation to the immune system is effected by having        at least two copies of an Aβ derived fragment or the analogue        covalently or non-covalently linked to a carrier molecule        capable of effecting presentation of multiple copies of        antigenic determinants.    -   15. The method according to any one of the preceding paragraphs,        variants b) or c), wherein the polyamino acid and T_(H) epitope        are attached to the polyhydroxypolymer by means of an amide        bond.    -   16. The method according to any one of the preceding paragraphs,        variants b) or c), wherein the the polyhydroxypolymer is a        polysaccharide.    -   17. The method according to any one of the preceding paragraphs,        wherein the analogue has been formulated with an adjuvant which        facilitates breaking of autotolerance to autoantigens.    -   18. The method according to any one of the preceding paragraphs,        wherein an effective amount of the analogue is administered to        the animal via a route selected from the parenteral route such        as the intracutaneous, the subcutaneous, and the intramuscular        routes; the peritoneal route; the oral route; the buccal route;        the sublinqual route; the epidural route; the spinal route; the        anal route; and the intracranial route.    -   19. The method according to paragraph 18, wherein the effective        amount is between 0.5 μg and 2,000 μg of the analogue.    -   20. The method according to any one of paragraphs 1-13, variant        a), wherein presentation of the analogue to the immune system is        effected by introducing nucleic acid(s) encoding the analogue        into the animal's cells and thereby obtaining in vivo expression        by the cells of the nucleic acid(s) introduced.    -   21. The method according to paragraph 20, wherein the nucleic        acid(s) introduced is/are selected from naked DNA, DNA        formulated with charged or uncharged lipids, DNA formulated in        liposomes, DNA included in a viral vector, DNA formulated with a        transfection-facilitating protein or polypeptide, DNA formulated        with a targeting protein or polypeptide, DNA formulated with        Calcium precipitating agents, DNA coupled to an inert carrier        molecule, DNA encapsulated in chitin or chitosan, and DNA        formulated with an adjuvant.    -   22. The method according to any one of paragraphs 18-21, which        includes at least one administration/introduction per year, such        as at least 2, at least 3, at least 4, at least 6, and at least        12 administrations/introductions.    -   23. The method according to any one of the preceding paragraphs        used for treating and/or preventing and/or ameliorating        Alzheimer's disease or other diseases and conditions        characterized by amyloid deposits, where APP or Aβ is        down-regulated to such an extent that the total amount of        amyloid is decreased or that the rate of amyloid formation is        reduced with clinical significance.    -   24. An analogue of APP or Aβ which is derived from an animal APP        or Aβ wherein is introduced a modification which has as a result        that immunization of the animal with the analogue induces        production of antibodies against the animal's autologous APP or        Aβ, and wherein the analogue is as defined in any one of        paragraphs 1-16.    -   25. An immunogenic composition comprising an immunogenically        effective amount of an analogue according to paragraph 24, the        composition further comprising a pharmaceutically and        immunologically acceptable carrier and/or vehicle and optionally        an adjuvant.    -   26. A nucleic acid fragment which encodes an analogue according        to paragraph 24.    -   27. A vector carrying the nucleic acid fragment according to        paragraph 26, such as a vector that is capable of autonomous        replication.    -   28. The vector according to paragraph 27 which is selected from        the group consisting of a plasmid, a phage, a cosmid, a        mini-chromosome, and a virus.    -   29. The vector according to paragraph 27 or 28, comprising, in        the 5′→3′ direction and in operable linkage, a promoter for        driving expression of the nucleic acid fragment according to        paragraph 26, optionally a nucleic acid sequence encoding a        leader peptide enabling secretion of or integration into the        membrane of the polypeptide fragment, the nucleic acid fragment        according to paragraph 26, and optionally a terminator.    -   30. The vector according to any one of paragraphs 27-29 which,        when introduced into a host cell, is capable or incapable of        being integrated in the host cell genome.    -   31. The vector according to paragraph 29 or 30, wherein the        promoter drives expression in a eukaryotic cell and/or in a        prokaryotic cell.    -   32. A transformed cell carrying the vector of any one of        paragraphs 27-31, such as a transformed cell which is capable of        replicating the nucleic acid fragment according to paragraph 26.    -   33. The transformed cell according to paragraph 32, which is a        microorganism selected from a bacterium, a yeast, a protozoan,        or a cell derived from a multicellular organism selected from a        fungus, an insect cell such as an S₂ or an SF cell, a plant        cell, and a mammalian cell.    -   34. The transformed cell according to paragraph 32 or 33, which        expresses the nucleic acid fragment according to paragraph 27,        such as a transformed cell, which secretes or carries on its        surface, the analogue according to paragraph 24.    -   35. The method according to any one of paragraphs 1-13, variant        a), wherein presentation to the immune system is effected by        administering a non-pathogenic microorganism or virus which is        carrying a nucleic acid fragment which encodes and expresses the        analogue.    -   36. A composition for inducing production of antibodies against        amyloid, the composition comprising        -   a nucleic acid fragment according to paragraph 26 or a            vector according to any one of paragraphs 27-31, and        -   a pharmaceutically and immunologically acceptable carrier            and/or vehicle and/or adjuvant.    -   37. A stable cell line which carries the vector according to any        one of paragraphs 7-31 and which expresses the nucleic acid        fragment according to paragraph 26, and which optionally        secretes or carries the analogue according to paragraph 24 on        its surface.

LIST OF REFERENCES

-   Brookmeyer, R.; Gray, S.; Kawas, C. (1998). Projections of    Alzheimer's Disease in the United States and the Public Health    Impact of Delaying Disease Onset. American Journal of Public Health,    88(9), 1337-1342.-   Buttini, M.; Orth, M.; Bellosta, S.; Akeefe, H.; Pitas, R. E.;    Wyss-Coray, T.; Mucke, L.; Mahley, R. W. (1999). Expression of Human    Apolipoprotein E3 or E4 in the Brains of Apoe−/− Mice:    Isoform-Specific Effects on Neurodegeneration. Journal of    Neuroscience, 19, 4867-4880.-   Clark, L. N.; Poorkaj, P.; Wszolek, Z.; Geschwind, D. H.;    Nasreddine, Z. S.; Miller, B.; Li, D.; Payami, H.; Awert, F.;    Markopoulou, K.; Andreadis, A.; D'Souza, I.; Lee, V. M.; Reed, L.;    Trojanowski, J. Q.; Zhukareva, V.; Bird, T.; Schellenberg, G.;    Wilhelmsen, K. C. (1998). Pathogenic Implications of Mutations in    the Tau Gene in Pallido-Ponto-Nigral Degeneration and Related    Neurodegenerative Disorders Linked to Chromosome 17. Proceedings of    the National Academy of Sciences U.S.A., 95(22), 13103-13107.-   Gupta, R. K. et. al. (1998), Dev Biol Stand. 92: 63-78.-   Hsiao K. et al. (1998) Transgenic mice expressing Alzheimer amyloid    precursor proteins”, Exp. Gerontol. 33 (7-8), 883-889-   Hutton, M.; Lendon, C. L.; Rizzu, P.; Baker, M.; Froelich, S.;    Houlden, H.; Pickering-Brown, S.; Chakraverty, S.; Isaacs, A.;    Grover, A.; Hackett, J.; Adamson, J.; Lincoln, S.; Dickson, D.;    Davies, P.; Petersen, R. C.; Stevens, M.; de Graaff, E.; Wauters,    E.; van Baren, J.; Hillebrand, M.; Joosse, M.; Kwon, J. M.; Nowotny,    P.; Che, L. K.; Norton, J.; Morris, J. C.; Reed, L. E.; Trojanowski,    J.; Basun, H.; Lannfelt, L.; Neystat, M.; Fahn, S.; Dark, F.;    Tannenberg, T.; Dodd, P.; Hayward, N.; Kwok, J. B. J.; Schofield, P.    R.; Andreadis, A.; Snowden, J.; Craufurd, D.; Neary, D.; Owen, F.;    Oostra, B. A.; Hardy, J.; Goate, A.; van Swieten, J.; Mann, D.;    Lynch, T.; Heutink, P. (1998). Association of Missense and    5′-Splice-Site Mutations in Tau with the Inherited Dementia FTDP-17.    Nature, 393, 702-705.-   Janus, C. et. al. (2000), Nature 408: 979-982.-   Kas, H. S. (1997) J Microencapsul 14: 689-711-   Leon, J.; Cheng, C. K.; Neumann, P. J. (1998). Alzheimer's Disease    Care: Costs and Potential Savings. Health Affairs, 17(6), 206-216.-   Lippa C. F. et al. (1998) Ab-42 deposition precedes other changes in    PS-1 Alzheimer's disease. Lancet 352, 1117-1118-   Luo, J.-J.; Wallace, W.; Riccioni, T.; Ingram, D. K.; Roth, G. S.;    Kusiak, J. W. (1999). Death of PC12 Cells and Hippocampal Neurons    Induced by Adenoviral-Mediated FAD Human Amyloid Precursor Protein    Gene Expression. Journal of Neuroscience Research, 55(5), 629-642.-   Naruse, S.; Thinakaran, G.; Luo, J.-J.; Kusiak, J. W.; Tomita, T.;    Iwatsubo, T.; Qian, X.; Ginty, D. D.; Price, D. L.; Borchelt, D. R.;    Wong, P. C.; Sisodia, S. S. (1998). Effects of PS1 Deficiency on    Membrane Protein Trafficking in Neurons. Neuron, 21(5), 1213-1231.-   National Institute on Aging Progress Report on Alzheimer's Disease,    1999, NIH Publication No. 99-4664.-   Pietrobon, P. J. (1995), Pharm Biotechnol. 6: 347-61Poorkaj, P.;    Bird, T. D.; Wijsman, E.; Nemens, E.; Garruto, R. M.; Anderson, L.;    Andreadis, A.; Wiederhold, W. C.; Raskind, M.; Schellenberg, G. D.    (1998). Tau Is a Candidate Gene for Chromosome 17 Frontotemporal    Dementia. Annals of Neurology, 43, 815-825.-   Schenk, D.; Barbour, R.; Dunn, W.; Gordon, G.; Grajeda, H.; Guido,    T.; Hu, K.; Huang, J.; Johnson-Wood, K.; Khan, K.; Kholodenko, D.;    Lee, M.; Liao, Z.; Lieberburg, I.; Motter, R.; Mutter, L.; Soriano,    F.; Shopp, G.; Vasquez, N.; Vandevert, C.; Walker, S.; Wogulis, M.;    Yednock, T.; Games, D.; Seubert, P. (1999). Immunization with A-beta    Attenuates Alzheimer's Disease-Like Pathology in the PDAPP Mouse.    Nature, 400(6740), 173-177.-   Shekunov, B. et. al. (1999), J. Crystal Growth 198/199: 1345-1351.-   Spillantini, M. G.; Murrell, J. R.; Goedert, M.; Farlow, M. R.;    Klug, A.; Ghetti, B. (1998). Mutation in the Tau Gene in Familial    Multiple System Tauopathy with Presenile Dementia. Proceedings of    the National Academy of Sciences U.S.A., 95(13), 7737-7741.-   Strittmatter, W. J.; Saunders, A. M.; Schmechel, D.; Pericak-Vance,    M.; Enghild, J.; Salvesen, G. S.; Roses, A. D. (1993).    Apolipoprotein E: High-Avidity Binding to Aβ and Increased Frequency    of Type 4 Allele in Late-Onset Familial Alzheimer Disease.    Proceedings of the National Academy of Sciences U.S.A.,    90,1977-1981.-   Vidal, R.; Frangione, B.; Rostagno, A.; Mead, S.; Revesz, T.; Plant,    G.; Ghiso, J. (1999). A Stop-Codon Mutation in the BRI Gene    Associated with Familial British Dementia. Nature, 399: 776-781.-   Zheng H. (1996) “Mice deficient for the amyloid precursor protein    gene. Ann. N Y Acad. Sci., 777, 421-426.-   York, P. (1999), PSTT 11: 430-440

1. A method for in vivo down-regulation of amyloid precursor protein(APP) or beta amyloid (Aβ) in an animal, the method comprising effectingpresentation to the animal's immune system of an immunogenicallyeffective amount of at least one analogue of APP or Aβ that incorporatesinto the same molecule a substantial fraction of B-cell epitopes of APPand/or Aβ so that the analogue reacts to the same extent as does APP orAβ with a polyclonal serum raised against APP or Aβ, and at least oneforeign T-helper epitope (T_(H) epitope) so that immunization of theanimal with the analogue induces production of antibodies against theanimal's autologous APP or Aβ, wherein the analogue a) is a polyaminoacid that contains the at least one foreign T_(H) epitope and adisrupted APP or Aβ sequence so that the analogue does not include anysubsequence of SEQ ID NO: 2 that binds productively to MHC class IImolecules initiating a T-cell response; and/or b) is a conjugatecomprising a polyhydroxypolymer backbone to which is separately coupleda polyamino acid as defined in a); and/or c) is a conjugate comprising apolyhydroxypolymer backbone to which is separately coupled 1) the atleast one foreign T_(H) epitope and 2) a disrupted sequence of APP or Aβas defined in a).
 2. The method according to claim 1, wherein the animalis a human being.
 3. The method according to claim 1, wherein at leastone first moiety is introduced which effects targeting of the analogueto an antigen presenting cell (APC) or a B-lymphocyte, and/or at leastone second moiety is introduced which stimulates the immune system,and/or at least one third moiety is introduced which optimizespresentation of the analogue to the immune system.
 4. The methodaccording to claim 3, wherein the first and/or the second and/or thethird moiety is/are attached as side groups by covalent or non-covalentbinding to suitable chemical groups in the APP or Aβ sequence.
 5. Themethod according to claim 1, wherein the analogue comprises a fusionpolypeptide.
 6. The method according to claim 1, wherein the analogueincludes duplication of at least one B-cell epitope of APP or Aβ and/orintroduction of a hapten.
 7. The method according to claim 1, whereinthe foreign T-cell epitope is immunodominant in the animal.
 8. Themethod according to claim 1, wherein the foreign T-cell epitope ispromiscuous.
 9. The method according to claim 8, wherein the promiscuousforeign T-cell epitope is selected from a natural promiscuous T-cellepitope and an artificial MHC-II binding peptide sequence.
 10. Themethod according to claim 8, wherein the natural T-cell epitope isselected from a Tetanus toxoid epitope, a diphtheria toxoid epitope, aninfluenza virus hemagluttinin epitope, and a P. falciparum CS epitope.11. The method according to claim 10, wherein the Tetanus toxoid epitopeis selected from P2 and P30.
 12. The method according to claim 1,wherein the analogue comprises B-cell epitopes which are not exposed tothe extracellular phase when present in a cell-bound form of theprecursor polypeptide Aβ.
 13. The method according to claim 1, whereinthe analogue lacks at least one B-cell epitope which is exposed to theextracellular phase when present in a cell-bound form of the precursorpolypeptide.
 14. The method according to claim 1, wherein the analoguecomprises at most 9 consecutive amino acids of SEQ ID NO:
 2. 15. Themethod according to claim 14, wherein the analogue comprises at most 8consecutive amino acids of SEQ ID NO:
 2. 16. The method according toclaim 14, wherein the analogue comprises at most 7 consecutive aminoacids of SEQ ID NO:
 2. 17. The method according to claim 14, wherein theanalogue comprises at most 6 consecutive amino acids of SEQ ID NO: 2.18. The method according to claim 14, wherein the analogue comprises atmost 5 consecutive amino acids of SEQ ID NO:
 2. 19. The method accordingto claim 14, wherein the analogue comprises at most 4 consecutive aminoacids of SEQ ID NO:
 2. 20. The method according to claim 14, wherein theanalogue comprises at most 3 consecutive amino acids of SEQ ID NO: 2.21. The method according to claim 14, wherein the analogue comprises atleast one subsequence of SEQ ID NO: 2 so that each such at least onesubsequence of SEQ ID NO: 2 independently consists of amino acidstretches selected from the group consisting of 9 consecutive aminoacids of SEQ ID NO: 2, 8 consecutive amino acids of SEQ ID NO: 2, 7consecutive amino acids of SEQ ID NO: 2, 6 consecutive amino acids ofSEQ ID NO: 2, 5 consecutive amino acids of SEQ ID NO: 2, 4 consecutiveamino acids of SEQ ID NO: 2, and 3 consecutive amino acids of SEQ ID NO:2.
 22. The method according to claim 14, wherein the consecutive aminoacids begin at an amino acid residue selected from the group consistingof residue 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683,684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697,698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711,712, 713, and
 714. 23. The method according to claim 1, whereinpresentation to the immune system is effected by having at least twocopies of an Aβ derived fragment or the analogue covalently ornon-covalently linked to a carrier molecule capable of effectingpresentation of multiple copies of antigenic determinants.
 24. Themethod according to claim 1, wherein the analogue is a conjugatecomprising a polyhydroxypolymer backbone to which is separately coupleda polyamino acid that contains the at least one foreign T_(H) epitopeand a disrupted APP or Aβ sequence so that the analogue does not includeany subsequence of SEQ ID NO: 2 that binds productively to MHC class IImolecules initiating a T-cell response and/or the analogue is aconjugate comprising a polyhydroxypolymer backbone to which isseparately coupled 1) the at least one foreign T_(H) epitope and 2) adisrupted sequence of APP or Aβ so that the analogue does not includeany subsequence of SEQ ID NO: 2 that binds productively to MHC class IImolecules initiating a T-cell response, wherein the polyamino acid andT_(H) epitope are attached to the polyhydroxypolymer by means of anamide bond.
 25. The method according to claim 1, wherein the analogue isa conjugate comprising a polyhydroxypolymer backbone to which isseparately coupled a polyamino acid that contains the at least oneforeign T_(H) epitope and a disrupted APP or Aβ sequence so that theanalogue does not include any subsequence of SEQ ID NO: 2 that bindsproductively to MHC class II molecules initiating a T-cell response,and/or the analogue is a conjugate comprising a polyhydroxypolymerbackbone to which is separately coupled 1) the at least one foreignT_(H) epitope and 2) a disrupted sequence of APP or Aβ so that theanalogue does not include any subsequence of SEQ ID NO: 2 that bindsproductively to MHC class II molecules initiating a T-cell response,wherein the polyhydroxypolymer is a polysaccharide.
 26. The methodaccording to claim 1, wherein the analogue has been formulated with anadjuvant which facilitates breaking of autotolerance to autoantigens.27. The method according to claim 1, wherein an effective amount of theanalogue is administered to the animal via a route selected from theparenteral route; the peritoneal route; the oral route; the buccalroute; the sublinqual route; the epidural route; the spinal route; theanal route; and the intracranial route.
 28. The method of claim 27,wherein the parenteral route is selected from the intracutaneous, thesubcutaneous, and the intramuscular routes.
 29. The method according toclaim 27, wherein the effective amount is between 0.5 μg and 2,000 μg ofthe analogue.
 30. The method according to claim 1, wherein the analogueis a polyamino acid that contains the at least one foreign T_(H) epitopeand a disrupted APP or Aβ sequence so that the analogue does not includeany subsequence of SEQ ID NO: 2 that binds productively to MHC class IImolecules initiating a T-cell response, wherein presentation of theanalogue to the immune system is effected by introducing nucleic acid(s)encoding the analogue into the animal's cells and thereby obtaining invivo expression by the cells of the nucleic acid(s) introduced.
 31. Themethod according to claim 30, wherein the nucleic acid(s) introducedis/are selected from naked DNA, DNA formulated with charged or unchargedlipids, DNA formulated in liposomes, DNA included in a viral vector, DNAformulated with a transfection-facilitating protein or polypeptide, DNAformulated with a targeting protein or polypeptide, DNA formulated withCalcium precipitating agents, DNA coupled to an inert carrier molecule,DNA encapsulated in chitin or chitosan, and DNA formulated with anadjuvant.
 32. The method according to claim 27, wherein the analogue isadministered at a frequency of at least one administration/introductionper year.
 33. The method according to claim 32, wherein the frequency ofadministration/introduction is selected from at least 2, at least 3, atleast 4, at least 6, and at least 12 administrations/introductions. 34.The method according to claim 1 used for treating and/or preventingand/or ameliorating Alzheimer's disease or other diseases and conditionscharacterized by amyloid deposits, where APP or Aβ is down-regulated tosuch an extent that the total amount of amyloid is decreased or that therate of amyloid formation is reduced with clinical significance.
 35. Ananalogue of APP or Aβ which is derived from an animal APP or Aβ whereinis introduced a modification which has as a result that immunization ofthe animal with the analogue induces production of antibodies againstthe animal's autologous APP or Aβ, and wherein the analogue is asdefined in claim
 1. 36. An immunogenic composition comprising animmunogenically effective amount of an analogue according to claim 35,the composition further comprising a pharmaceutically andimmunologically acceptable carrier and/or vehicle and optionally anadjuvant.
 37. A nucleic acid fragment which encodes an analogueaccording to claim
 35. 38. A vector carrying the nucleic acid fragmentaccording to claim 37, such as a vector that is capable of autonomousreplication.
 39. The vector according to claim 38 which is selected fromthe group consisting of a plasmid, a phage, a cosmid, a mini-chromosome,and a virus.
 40. The vector according to claim 38, comprising, in the5′→3′ direction and in operable linkage, a promoter for drivingexpression of the nucleic acid fragment which encodes an analogue of APPor Aβ which is derived from an animal APP or Aβ wherein is introduced amodification which has as a result that immunization of the animal withthe analogue induces production of antibodies against the animal'sautologous APP or AP, optionally a nucleic acid sequence encoding aleader peptide enabling secretion of or integration into the membrane ofthe polypeptide fragment, the nucleic acid fragment which encodes ananalogue of APP or Aβ which is derived from an animal APP or Aβ whereinis introduced a modification which has as a result that immunization ofthe animal with the analogue induces production of antibodies againstthe animal's autologous APP or Aβ, and wherein the analogue is: a) is apolyamino acid that contains the at least one foreign T_(H) epitope anda disrupted APP or Aβ sequence so that the analogue does not include anysubsequence of SEQ ID NO: 2 that binds productively to MHC class IImolecules initiating a T-cell response; and/or b) is a conjugatecomprising a polyhydroxypolymer backbone to which is separately coupleda polyamino acid as defined in a); and/or c) is a conjugate comprising apolyhydroxypolymer backbone to which is separately coupled 1) the atleast one foreign T_(H) epitope and 2) a disrupted sequence of APP or Aβas defined in a); and optionally a terminator.
 41. The vector accordingto claim 38 which, when introduced into a host cell, is capable orincapable of being integrated in the host cell genome.
 42. The vectoraccording to claim 40, wherein the promoter drives expression in aeukaryotic cell and/or in a prokaryotic cell.
 43. A transformed cellcarrying the vector of claim
 38. 44. The transformed cell of claim 43,wherein the transformed cell is capable of replicating a nucleic acidfragment which encodes an analogue of APP or Aβ which is derived from ananimal APP or Aβ wherein is introduced a modification which has as aresult that immunization of the animal with the analogue inducesproduction of antibodies against the animal's autologous APP or Aβ. 45.The transformed cell according to claim 43, which is a microorganismselected from a bacterium, a yeast, a protozoan, or a cell derived froma multicellular organism selected from a fungus, an insect cell, a plantcell, and a mammalian cell.
 46. The transformed cell of claim 45,wherein the insect cell is selected from an S₂ and an SF cell.
 47. Thetransformed cell according to claim 43, which expresses nucleic acidfragment which encodes an analogue of APP or Aβ which is derived from ananimal APP or Aβ wherein is introduced a modification which has as aresult that immunization of the animal with the analogue inducesproduction of antibodies against the animal's autologous APP or Aβ. 48.The transformed cell according to claim 43, wherein the transformed cellsecretes or carries on its surface an analogue of APP or Aβ which isderived from an animal APP or Aβ wherein is introduced a modificationwhich has as a result that immunization of the animal with the analogueinduces production of antibodies against the animal's autologous APP orAβ.
 49. The method according to claim 1, wherein the analogue is apolyamino acid that contains the at least one foreign T_(H) epitope anda disrupted APP or Aβ sequence so that the analogue does not include anysubsequence of SEQ ID NO: 2 that binds productively to MHC class IImolecules initiating a T-cell response, wherein presentation to theimmune system is effected by administering a non-pathogenicmicroorganism or virus which is carrying a nucleic acid fragment whichencodes and expresses the analogue.
 50. A composition for inducingproduction of antibodies against amyloid, the composition comprising anucleic acid fragment which encodes an analogue of APP or Aβ which isderived from an animal APP or Aβ wherein is introduced a modificationwhich has as a result that immunization of the animal with the analogueinduces production of antibodies against the animal's autologous APP orAβ or a vector according to claim 27, and a pharmaceutically andimmunologically acceptable carrier and/or vehicle and/or adjuvant.
 51. Astable cell line which carries the vector carrying a nucleic acidfragment which encodes an analogue according to claim 35, such as avector that is capable of autonomous replication and which expresses thenucleic acid fragment which encodes an analogue according to claim 35,and which optionally secretes or carries the analogue according to claim35 on its surface.